VZV gene, mutant VZV and immunogenic compositions

ABSTRACT

The invention provides for a novel Varicella-Zoster Virus gene, mutant Varicella-Zoster Virus and immunogenic compositions based on such novel genes and mutant VZV. Also provided are proteins, diagnostic assays and methods of producing reconstructed VZV.

This is a continuation of application Ser. No. 08/235,406 filed Apr. 28,1994, now abandoned.

TECHNICAL FIELD

The invention relates to the identification of a novel Varicella-ZosterVirus (hereinafter to be referred to as VZV) gene. It also relates tomethods of making and to novel immunogenic compositions comprising VZV,modified by the modification or deletion of this novel gene and tomethods inducing an immune response in a host organism by administrationof such immunogenic compositions.

BACKGROUND

Varicella-Zoster Virus, a member of the herpes virus family, causeschicken pox as a primary infection and shingles as a secondaryinfection. Chicken pox is a common disease of children that is highlycontagious but usually not life threatening. In adults chicken pox canpose a more serious medical threat. Shingles occurs after primaryinfection with the vaccine or the wild type virus. After the primaryinfection, the virus travels to the trigeminal or thoracic (commonlyT3-L2) ganglia and lies dormant. Upon reactivation, the virus affectsthe dermatome supplied by the corresponding ganglia. Both of thesediseases are widespread, with 3-4 million varicella and 850,000 zostercases in the U.S. per year, and costs $700 million per year in the U.S.in treatment and lost work costs.

Chemotherapy is available to combat VZV infections. Acyclovirchemotherapy halts the progression of disease by acting as a substratefor herpes pyrimidine deoxyribonucleotide kinase. The kinasephosphorylates Acyclovir, and the phosphorylated Acyclovir is thenincorporated into viral DNA, which ultimately leads to DNA chaintermination. Although chemotherapy is effective in treating infectedpatients with varicella or zoster symptoms it does not preventoccurrence of either disease.

An alternative post-infection treatment, VZV-specific immunoglobulin, isavailable, in addition to chemotherapy, to treat VZV infections.However, this treatment is expensive, affords only a narrow window oftherapeutic use and must be quality controlled for HIV contamination.

A live, attenuated vaccine made of VZV derived from the Oka strain ofthat organism has been developed by Takahashi, M., et al., Lancet 1974;2:1288-90. The vaccine was developed by multiple passage of the virusthrough a mammalian cell line. It protects against chicken pox inhumans. However, zoster infections resulting from Oka replication havebeen documented in humans vaccinated with the Oka vaccine.Unfortunately, no other vaccines against VZV are currently marketed.

Progress toward developing a VZV vaccine has been stymied because nopractical animal model exists for determining vaccination and immuneresponses and because only low viral yields in culture are currentlyavailable. Guinea pigs, and perhaps marmosets, can be used as limitedmodels for VZV studies. Infection can be documented in these animals,although latency and reactivation of the virus has yet to beestablished. One 6 month old gorilla has been shown to be infected witha VZV indistinguishable from a human strain. Even though the diseaseclosely resembled that of human varicella, the gorilla is an impracticaldisease model for obvious socio-economic reasons. Although cultureyields have been improved somewhat by growing attenuated vaccines inmonolayers (see for example PCT Patent Application WO93/24616 publishedDec. 9, 1993), higher yields would be even more advantageous.Additionally, deletions in the VZV genome to produce attenuated strainsof VZV which would make good candidates for live vaccines have yieldedvarying results. For example, deletion of the gene encodingribonucleotide reductase slowed viral growth and increased Acyclovirsensitivity. In contrast, prevention of VZV thymidylate synthetase geneexpression failed to affect viral growth rates.

As noted previously, VZV is a member of the herpesvirus family. As such,the 120 kilobase (kb) VZV genome shares some structural homology toherpes simplex virus. Both viruses store their virion DNA as a linearmolecule and replicate using circular DNA molecules. The linear order ofgenes within the each DNA molecule is similar for most genes in bothherpes simplex virus and VZV. Each genome contains two unique sequences.U_(L) (standing for "unique long") comprises about 100 kb and U_(S)(standing for "unique short") comprises about 5 kb. Each U_(L) sequenceis flanked by a terminal repeat sequence, i.e., a TR_(L) (or terminalrepeat of unique long), and an internal repeat sequence, i.e., an IR_(L)(internal repeat of unique long). Likewise, each U_(S) sequence isflanked by a terminal repeat sequence, TR_(S), and an internal repeatsequence, IR_(S).

However, VZV and herpes simplex virus display numerous differences intheir genomic structure as well. The herpes simplex virus genomecontains an extra set of inverted repeats called the "a" sequence, inaddition to TR_(L), IR_(L), TR_(S) and IR_(S) sequences. The a sequencesare located at both genomic termini, as well as at the junction of the Land S components. The VZV TR_(L) and IR_(L) sequences are very short (88base pairs) compared to the herpes simplex virus TR_(L) and IR_(L)sequences (8000 base pairs). Another difference is that there is anorigin of DNA replication at the approximate center of the U_(L)sequence in herpes simplex virus. There is no such structure in VZV. Thefunctional and evolutionary significance of the differences between therepeat regions flanking the UL region in herpes simplex virus and therepeat regions flanking the U_(L) region in VZV is unknown.

Four different genomic isomers, composed of Par (Parental) and inverted(Inv) forms of the U_(L) and U_(S) repeat sequences, exist in the VZVgenome: U_(L) -Par/U_(S) -Par, U_(L) -Par/U_(S) -Inv, U_(L) -Inv/U_(S)-Par, and U_(L) -Inv/U_(S) -Inv. The four isomers are not randomlydistributed. Two isomers (U_(L) -Par/U_(S) -Par and U_(L) -Par/U_(S)-Inv) account for 95% of the packaged DNA and the remaining two isomers(U_(L) Inv/U_(S) -Par and U_(L) -Inv/U_(S) -Inv) account for 5% of thepackaged DNA, as shown in FIG. 1.

The VZV genome contains 80 possible open reading frames (hereinafterabbreviated as ORF or ORFs), although fewer genes, approximately 70, arethought to encode gene products used in the viral replication cycle. TheORF's of VZV are based on ORF criteria from Davison, A. J. and Scott, J.E., Journal of General Virology (1983); 64:1811-184 (ORFs with amethionine initiation site and at least 150 amino acids or ORFs with aTATA box, no overlap with other ORFs and good codon usage). Little isknown about the function of the approximately twenty known VZV geneproducts. The novel gene identified herein was not detected by Davison &Scott type ORF criteria.

Some of the VZV gene products lacking a demonstrated function shownucleic acid or amino acid sequence homology to herpes simplex virusgenes of known function. As used herein, "homolog" or "homologous"refers solely to nucleic acid or protein sequence homology between twosequences from different organisms, and does not encompass anyfunctional similarity. About 62 of the known VZV genes have homologs inthe herpes simplex virus genome. Five known VZV genes have no homologsin the herpes simplex virus genome. Homologs are traditionallydetermined using computer sequence analysis methods or using nucleotideprobing of nucleotide sequences, methods that require a level ofsequence homology sufficient to allow recognition of homologs abovebackground. The novel ORFS/L gene disclosed herein for the first timehad not been previously detected using either of the traditionalmethods. Surprisingly, it has been found that this novel gene isactually a positional homolog of the γ₁ 34.5 gene of herpes simplex. Itapparently previously escaped detection because it lacks a typical TATAconsensus element upstream of its open reading frame, it has an unusualand unexpected gene structure and it is located in an unexpectedlocation of the VZV genome.

RELEVANT BACKGROUND LITERATURE

The role of herpes simplex virus γ₁ 34.5 gene in neuro-virulence andgrowth was disclosed in Science; 250:1262-1266 (1990), "Mapping ofHerpes Simplex Virus-1 Nurovirulence to γ₁ 34.5, a gene Nonessential forGrowth in Culture" by J. Chou, E. R. Kern, R. J. Whitley, and B.Roizman.

The existence of a herpes simplex virus gene product near the internalrepeat region of herpes simplex virus genome was disclosed in theJournal of Virology. 57:629-637 (1986), "The Terminal a Sequence of theHerpes Simplex Virus genome contains the Promoter of a Gene Located inthe Repeat Sequences of the L Component" by J. Chou and B. Roizman.

It has been disclosed that herpes simplex viral mutants, lacking afunctional γ₁ 34.5 gene, expressed early proteins, but viral DNAsynthesis resulted in the cessation of viral protein synthesis. Proc.Natl. Acad. of Sci. USA. 89:3266-3270 (1992), "The γ₁ 34.5 gene ofherpes simplex virus 1 precludes . . . " by J. Chou and B. Roizman.

It has been disclosed that the Oka strain of VZV can be reconstructedusing overlapping cosmid clones and that mutants, lacking a functionalthymidylate synthetase gene, grew at a rate similar to the wild typevirus. Pro. Natl. Acad. of Sci. USA. 90:7376-7380 (1993), "Generation ofVaricella-Zoster Virus (VZV) and viral mutants from cosmid DNAs: . . . "by J. L. Cohen and K. E. Seidel.

The introduction of a stop codon into the thymidylate synthetase generesulted in the lack of thymidylate synthetase protein expressionwithout affecting viral growth rates or Acyclovir sensitivity, asdisclosed in XVIII International Herpesvirus Workshop Abstract 1993,"Generation of Varicella-Zoster Virus and Viral Mutants from Cosmic DNA;Thymidylate Synthetase is Not Essential for Replication In Vitro" byJeffrey I. Cohen and Karen E. Seidel.

For a general review of VZV see Journal of Virology 72:475-486 (1991),"Varicella-Zoster Virus" by A. J. Davidson.

The DNA sequence and open reading frames of VZV are disclosed in Journalof General Virology. 67:1759-1816 (1986), "The Complete DNA Sequence ofVaricella-Zoster Virus" by A. J. Davison and J. E. Scott.

A bovine Herpes virus gene that is formed during the circularization ofa bovine Herpes virus genome was disclosed in the Journal of Virology;67:1328-1333 (1993), "Immediate-Early Transcription over CovalentlyJoined Genome Ends of Bovine Herpesvirus-1: the circ Gene" by C.Fraefel, et al.

The deletion of VZV ORF's for ribonucleotide reductase slowed viralgrowth rate, increased Acyclovir sensitivity and reduced plaque size, asdisclosed in XVIII International Herpes Workshop Abstract 1993,"Production and Characterization of a VZV Mutant Lacking the LargeSubunit of Ribonucleotide Reductase" by T. C. Heineman and J. I. Cohen.

The immune response of elderly individuals to the Oka vaccine isdiscussed in The Journal of Infectious Diseases; 166:253-9 (1992),"Immune Response of Elderly Individuals to a Live Attenuated VaricellaVaccine" by Levin, M. J., et al.

The hairless guinea pig model is disclosed in The Journal of InfectiousDiseases; 163:746-751 (1991), "Varicella in Hairless Guinea Pigs" byMyles, M. G., et al.

Infection of a gorilla by varicella was disclosed in Journal of MedicalVirology, 23:317-322 (1987), "Varicella in Gorilla" by Myer, M. G. etal.

Lastly, U.S. Pat. No. 4,686,101 (1987); U.S. Pat. No. 4,769,239 (1988),U.S. Pat. No. 4,812,559 (1989) and U.S. Pat. No. 4,952,674 (1990) issuedto Ellis and Keller disclose the isolation and cloning of the gCglycoprotein of VZV and their use in vaccines against VZV.

DESCRIPTION OF THE FIGURES

FIGS. 1a-d illustrates the four different isomers of the VZV genome. Theisomers are packaged in capsids and differ in the orientation of theirunique region sequences. The U_(L) -Par isomers comprise 95% of thepackaged DNA. The U_(L) -Inv isomers comprise only 5% of the packagedDNA. The abbreviations associated with each isomer are described in thebackground section.

FIG. 2 illustrates the general arrangement of three separate readingframes that potentially comprise the ORFS/L gene in Dumas and Oka. Thestriped region indicates the first open reading frame of the ORFS/Lgene. The dark areas, from left to right, represent the second and thirdreading frames of the ORFS/L gene. The U_(L) -Par isomer in its linearform fails to create a ORFS/L reading frame. The U_(L) -Inv isomercreates a complete ORFS/L reading frame at the internal repeat region.The U_(L) -Par isomer and its concatamic form generates an ORFS/L gene.The circular form of the U_(L) -Par isomer also generates a ORFS/L gene.

FIG. 3 illustrates one possible gene structure of ORFS/L reading frames(based on U_(L) -Inv) used for isolating the ORFS/L gene with thepolymerase chain reaction and primers as discussed in Example 2.

FIG. 4 illustrates the 4 overlapping segments of VZV genome cloned intocosmids made in Example 5 and the restriction sites used in thosecosmids. VZV genomic DNA was cut into 4 fragments as shown usingrestriction enzymes Fsp I, Spe I and Pme I. The fragments were clonedinto cosmids as discussed in Example 5.

FIG. 5 illustrates the DNA sequence of the ORFS/L gene from Dumas andthe corresponding amino acid sequence of the ORFS/L protein encoded bythe nucleotide sequences. The DNA sequence comprises either the firstand second reading frames or the first and third reading frames asillustrated in FIG. 2. The second and third reading frames are identicalin Dumas. The location of the reading frame junction between the secondand first ORF is indicated by the slash "/" mark in FIG. 5.

FIGS. 6a and 6b illustrate the DNA sequence of the ORFS/L gene from Okaand the corresponding amino acid sequence of the ORFS/L proteins encodedby the nucleotide sequences. FIG. 6a is the nucleotide and correspondingamino acid sequence of the PCR clone p S/L C3 generated from Oka. FIG.6b is the nucleotide and corresponding amino acid sequence of the firstORF from the ORFS/L gene generated from a plasmids containing Okagenomic DNA, pV4L. Amino acid "Xxx" denotes one of the following aminoacids proline, threonine, serine, and alanine. The second and thirdreading frames are nearly identical in Oka and are not shown in FIG. 6b(they are listed as SEQ ID NO:15 and SEQ ID NO:16, respectively). Thelocation of the reading frame junction between the second and first ORFis indicated by the slash "/" mark in FIG. 6a.

FIGS. 7a-c illustrates a comparison of the following ORFS/L gene DNASequences: ORFS/L gene (Dumas) (SEQ ID NO:9), m13 S/L C3 from the ORFS/Lgene of Oka (SEQ ID NO:11), pV4L from the ORFS/L gene from genomic OkaDNA (SEQ ID NO:13), pV21J from the ORFS/L gene from genomic Oka DNA (SEQID NO:15), pV21S from the ORFS/L gene from genomic Oka DNA (SEQ IDNO:16) and the complete ORFS/L gene from Oka genomic DNA (SEQ ID NO:17).When used in nucleotide sequences, as shown herein, for example in theFIGS. and sequence listing, "V" denotes A or C or G and not T/U; "H"denotes A or C or T/U and not G; and "Y" denotes C or T/U.

FIG. 8 illustrates the expression of various Oka ORFS/L genes andconstructs as discussed in Examples 1, 2, 3 and 5.

SUMMARY OF THE INVENTION

The present invention provides a novel VZV gene, termed Open ReadingFrame S/L (abbreviated herein as ORFS/L), not heretofore recognized orknown in the art. The new ORFS/L was originally found in the Dumasstrain of VZV and comprises two combinations of three previously unknownand unidentified ORFs near the terminal and internal repeat regions ofthe VZV genome. (The gene's name, ORFS/L, is derived from theobservation that the S and L components must be joined to create theopen reading frame.) The new ORFS/L has also been identified in the Okastrain of VZV. Prior to the invention, the location and sequence of theORFS/L eluded detection. The ORFS/L was refractory to discovery due tolack of consensus initiation and polyadenylation signals, its unexpectedgene structure and its unusual location in the VZV genome. The inventionalso provides novel ORFS/L proteins encoded by the gene.

In another aspect the invention provides mutant VZV comprising all or aportion of the VZV genome having a mutation in the ORFS/L gene.

In another aspect the invention provides immunizing compositionscomprising such mutant viruses.

In yet another aspect, the invention comprises a method of making suchimmunizing compositions and also provides other diagnostic andtherapeutic uses for the gene and its protein product.

Most desirably, mutant viruses and immunogenic compositions are providedthat comprise VZV attenuated with respect to both varicella primaryinfections and zoster secondary infections. The mutant VZV of thisinvention comprises VZV in which the gene of the newly identified openreading frames ORFS/L gene are deleted, modified by nucleic acidsubstitution or by partial deletion of one or more nucleic acids, or byinsertion of a codon that terminates translation of the gene. The mutantVZV of this invention may be also used with a deletion or mutation ofother non-essential and essential VZV genes. As used here, "mutant VZV"means VZV containing at least a mutation in the ORFS/L gene ("mutation"as used in this specification is defined in the Detailed Description).Such compositions will retain the immunogenic character of VZV but willbe unable to exhibit neurovirulence characteristic of the virus.

In accordance with the invention, the ORFS/L gene sequence is altered tocreate mutant VZV and compositions having immunogenic properties.Alterations include deletion of the entire gene, part of the gene, sitespecific mutations, and introduction of a translation termination signalinto the gene. It is anticipated that the VZV containing the alteredgene will lack its normal neuro-virulence but retain its immunogeniccharacter. If the ORFS/L gene is modified in a VZV strain alreadyattenuated for varicella, such as the Oka strain currently approved as avaccine, it is anticipated that a virus with attenuated characteristicsfor both varicella and zoster will result. Immunogenic compositionscontaining such modified strains may result in improved vaccinecompositions in which the possibility of causing a latent zosterinfection by administration of the VZV vaccine is eliminated.

In addition, the novel ORFS/L gene and its protein product(s) can beused in the study of programmed cell death (apoptosis) and in thepreparation of diagnostic assays for VZV infection, as discussed morefully below.

DETAILED DESCRIPTION

A. Introduction

The invention provides a novel VZV gene, termed ORFS/L, not heretoforerecognized or known in the art. The invention also provides mutant VZVcharacterized by having at least one mutation in the ORFS/L gene, amutation which results in modified or altered production of ORFS/Lprotein. The invention also provides immunization compositions andmethods comprising the mutant VZV of the invention and also providesother diagnostic and therapeutic uses for the gene and its proteinproduct. The new ORFS/L gene was originally found in the Dumas strain ofVZV and comprises two combinations of three previously unknown ORFs nearthe terminal and internal repeat regions of the VZV genome. The newORFS/L gene has also been identified in the Oka strain of VZV. Detailsof its sequence and structural characteristics are provided in theExamples below. Briefly, the ORFS/L gene comprises VZV (Dumas) genomicDNA corresponding to a sequence of a first ORF from nucleotides 1through 562, a second ORF from nucleotides 104,925 through 105,125 and athird ORF from nucleotides 124,772 through 124,884. The ORFS/L genenucleotide sequence from Dumas encodes a 224 amino acid protein,starting at the first ATG, having a predicted molecular weight for themature protein of about 24,265 daltons.

Most desirably, mutant VZV and VZV immunogenic compositions includingmutant VZV are provided that comprise VZV attenuated with respect toboth varicella primary infections and zoster secondary infections. Themutant VZV and VZV immunogenic compositions of this invention compriseVZV in which the gene and in most instances the gene product of ORFS/Lgene are modified by deletion, nucleic acid substitution or deletion, orby insertion of a codon that stops translation of the gene, in order todetrimentally affect neurovirulence while retaining the immunogeniccharacter of VZV.

Thus, one aspect of this invention includes isolated DNA andcorresponding RNA sequences that encode the ORFS/L proteins. As usedherein, "isolated" means substantially free from other nucleotide orpolypeptide sequences with which the subject nucleotide sequence orpolypeptide sequence is typically found in its native, i.e., endogenous,state. In another aspect, the invention comprises isolated ORFS/Lprotein.

Another aspect of this invention includes mutant VZV having at least onemutation in the ORFS/L gene. The identification of the ORFS/L genepermits the introduction of defined mutations in the VZV genome. Themodified VZV compositions of this invention are characterized by genemutation or mutations that produce no substantial disease states,diminish the likelihood of reactivation that leads to shingles,stimulate the immune system and replicate in cells of non-neuronaltissue origin.

Yet another aspect of this invention includes diagnostic assays for thedetection of VZV strain variants. In brief, such diagnostic assaysinclude the use of ORFS/L fragments as primers for amplifying ORFS/Lrelated nucleic acids in a polymerase chain reaction (PCR), and the useof ORFS/L genes modified with a unique restriction site to act asvaccine markers.

It is anticipated that the invention will enable the production ofvaccines that offer advantages over the current VZV vaccine, which cancause shingles post vaccine infection. Due to the unique nature of theORFS/L gene, the likelihood of developing shingles post administrationof the modified VZV immunogenic compositions of the present invention isexpected to be much less than the likelihood of developing shingles fromcurrently used vaccines. Vaccines produced in accordance with theinvention will have a second advantage over cell passage vaccines forVZV because the degree of attenuation achieved can be predicted andmaintained throughout production and the likelihood of reversion issignificantly less in the mutant VZV and VZV immunogenic compositions ofthe present invention as compared with the currently used cell passageVZV vaccine. More specifically, modification or alteration of the novelORFS/L gene of the present invention results in the introduction ofspecific, desired and chosen changes in the VZV genome that are notpossible using the cell passage vaccines, which contain onlynonselective mutations. Such mutation allows for easier identificationof individuals infected with wild type VZV and therefore has usefuldiagnostic applications. More importantly for vaccine productionhowever, it also enables precise measurement of the degree ofattenuation introduced by different mutations in the ORSF/L gene.

The mutant VZVs of the present invention stimulate the immune systembecause sufficient amounts of VZV glycoprotein, which form the basis ofthe immune system's response to viral infection, are retained in thecomposition to stimulate an immune response. Mutant VZV compositions ofthe present invention contain these glycoproteins, which aresufficiently similar to the wild type glycoproteins, to stimulate animmune response because regions of VZV genome encoding suchglycoproteins that are known to be important antigens are not altered inthe mutant VZV compositions of the present invention.

Mutant VZVs of the present invention comprising mutations,substitutions, additions, deletions or stop codon introductions in theORFS/L gene still permit efficient viral replication in cells ofnon-neuronal tissue origin. Approximately, 14 DNA metabolism enzymes arepresent in the VZV genome. In the mutant VZVs of this invention, theseenzymes remain sufficiently similar to their counterparts in the wildtype virus so as to maintain efficient replication in non-neuronaltissues. The ORFS/L gene was selected for modification, in part, becauseit bears no sequence homology to DNA metabolism enzymes, which wouldpotentially affect viral propagation in cells of non-neuronal tissueorigin. Accordingly, the mutant VZVs of the present invention retain theimmunogenicity of wild type VZV.

B. Mutant VZVs and Immunogenic Compositions

The ORFS/L gene comprises ORFS/L protein coding regions, and ORFS/Lnoncoding regions. Protein coding regions are delineated by the aminoacid sequence encoded by ORFS/L gene. The noncoding regions aredelineated by either the transcript size of the ORFS/L gene, regions ofhomology surrounding ORFS/L gene reading frames, or sequences in thenoncoding regions which control the initiation and termination oftranscription. As used herein, the term "ORFS/L gene" refers to anORFS/L gene from any VZV strain, unless as a specific strain isenumerated. For example, "ORFS/L (Dumas)" and "ORFS/L (Oka)" refer tothe ORFS/L of the Dumas and Oka strains, respectively. Also as usedherein, the term "ORFS/L gene" encompasses the DNA and RNA version ofthe gene, single stranded or double stranded, and in the sense orantisense orientation. Different strains of VZV ORFS/L genes, whetherfrom recombinant sources, synthesized, or naturally occurring, that are90% homologous to either the Dumas or Oka ORFS/L gene (as described inFIGS. 5, 6 and/or 7 and SEQ ID NO:9-17) are also considered to be ORFS/Lgenes or fragments thereof, as defined herein.

Mutant VZVs comprise VZV characterized as containing at least onemutation in the ORFS/L gene that alters the function or the level ofexpression of the ORFS/L gene product. Such a mutation can include theintroduction of a termination signal in the ORFS/L gene, the deletion ofall or a part of the coding or noncoding sequence, the introduction ofone or more extra nucleotide(s) that changes the reading frame of thegene, and point mutations or site specific deletion or substitution ofspecific nucleotides using techniques already known in the art.Introduction of a termination signal is accomplished, for instance,using stop sequences in single or multiple reading frames. Mutation inthe coding region of the ORFS/L gene comprising single or multiplesubstitution, insertion or deletion will alter the function of theORFS/L protein by directly modifying the polypeptide sequence encoded bythe ORFS/L nucleotide sequence. Mutations can also be made that do notchange the polypeptide sequence but only change the nucleic acidsequence of the ORFS/L gene or substitute degenerate codons for thenative codons, in order to change the codon frequency or introduce arestriction enzyme site. The ORFS/L gene can also be modified bymutation of the transcriptional control elements of the gene in order toalter the level of expression of the ORFS/L gene product. Suchmodification can be accomplished in the foregoing manner usingsubstitutions, insertions or deletions in the nucleotide sequence. Asused here, "mutation" includes any of the foregoing modifications ingene structure disclosed herein, including single or multiple pointmutations or site specific substitutions or deletions, single ormultiple insertions or additions in the nucleotide sequence, deletionsor substitution of all or a substantial part of the nucleotide sequenceor single or multiple substitution or deletion of part of the nucleotidesequence of the ORFS/L gene.

With respect to mutations that comprise deletions in the gene, thelength of the deletion, whether single or multiple, will preferablytotal at least 30 bases in length, more preferably at least 75 bases,and most preferably at least 300 bases. Deletions of nucleotides thatencode amino acids conserved between VZV strains are preferred. Theportion of the ORFS/L gene remaining after deletion is one embodiment ofa fragment of the ORFS/L gene discussed herein. Also, preferred aredeletions in the coding region of the ORFS/L gene nucleic acid sequencethat comprise deletions of at least one amino acid selected from thegroup consisting of Glutamine, Asparagine, Arginine, Lysine, andProline. More preferably, the deleted ORFS/L nucleotides encode at leasttwo amino acids selected from that group. For example, a preferredmutant VZV composition of the present invention might include VZV havinga nucleic acid sequence deletion from the ORFS/L gene encoding oneGlutamine and one Arginine. Most preferable are mutant VZV compositionscomprising VZV characterized by having deletions comprising a portion ofthe ORFS/L nucleotide sequence that encode at least three prolineresidues.

With respect to mutations that comprise one or more alterations ordeletions in the coding region of the ORFS/L gene, mutations that resultin an amino acid sequence that is at least 80% homologous to the wildtype ORFS/L amino acid sequence are preferred, and mutations that resultin amino acid sequences that are 90% homologous are especiallypreferred. Mutant VZV compositions in which the ORFS/L gene containsmutations in that portion of the gene located in the unique long regionof the VZV genome are especially preferred.

In some embodiments, mutations of the ORFS/L gene will alter the abilityof the mutant VZV to grow in cells of neuronal tissue origin bydecreasing or preventing propagation in cells of neuronal tissue origin.Preferably an ORFS/L gene mutation will reduce viral growth in cells ofneuronal tissue origin by at least 20%, more preferably by at least 50%and most preferably by at least 75%. The test for attenuatedneurovirulence and growth in cells from non-neuronal tissue is describedin Example 10.

Since a mutation introduced into the ORFS/L gene could result in reducedproduction of the virus, it may be required that the gene product of theORFS/L gene be supplied in trans to achieve good replication of themodified VZV in culture for commercial production. Should ORFS/L proveto be essential for growth of commercial quantities of VZV in culture,it will be desirable to derive a cell line that can support replicationof the recombinant virus. The ORFS/L coding region, will be placed undertranscriptional control of a promoter such as the CMV (cytomeglovirus)major immediate early promoter, the SV40 early promoter or some otherviral or cellular promoter that generates adequate levels of expression,as discussed herein. A cell line, such as VERO, will be cotransfectedwith a plasmid containing the promoter driven ORFS/L gene and a plasmidcontaining a drug resistance gene. The cells will be selected for growthin the presence of the appropriate antibiotic. For example, if the drugresistance gene is neo, the cells will be selected for growth in thepresence of G418. Resistant cell lines will then be tested for theirability to support the growth of the recombinant virus by cotransfectingthe overlapping cosmids containing the appropriate mutation anddetermining whether infectious virus was formed. These cell lines willthen be used to generate large amounts of the recombinant virus. Moresubtle changes in the ORFS/L gene, if required to facilitate vaccineproduction, are attained using alterations of the transcriptionpromotion region.

A mutant VZV can be made by isolating the native ORFS/L gene in a wildtype virus strain or any recombinant VZV and incorporating the desiredmutation into the ORFS/L gene as discussed herein. Mutant VZV can alsobe produced using VZV with additional modifications in other essentialand non-essential genes, such as, but not limited to, the uracil-DNAglycosylase or thymidylate synthetase genes. The modified gene can thenbe reincorporated into a viral genome or virus using techniques known inthe art, such as homologous recombination using flanking sequences; orthe modified gene can be reincorporated into a viral genome or virususing cosmid techniques discussed herein and in the art. Because thesecond and third ORF's of the ORFS/L gene are located in the IR_(S)/IR_(L) and TRs repeat sequences, respectively, any intended mutation ofor within these regions will require that both copies of the sequencepresent in the wild type virus are deleted or altered. Alternatively, amutant VZV may be made comprising a modification of the first ORF suchthat duplicative mutation is unnecessary in the second and third ORF's.Because 474 nucleotides of the first ORF (corresponding to nucleotidesin the region of 1 through 562 (first ORF) of the VZV (Dumas) genomicsequence encoding ORFS/L) are not repeated, mutations in the first ORFof the sequence the ORFS/L gene are preferred.

Alternatively, whole wild type VZV need not be used as startingmaterial. Defective VZV vectors can be used. Such vectors may beconstructed using known techniques. Exemplary defective vectors, whichtypically contain DNA or RNA sequences including the viral packagingsite, the origin of replication, a promoter sequence to directtranscription and a translation limitation sequence, a polyadenylationsequence to terminate transcription and a selectable marker or reportersequence to permit selection of the vector. Such defective vectorsconstructed from Herpes simplex 1 sequences are described in PCT PatentPublications WO90/09441 published Aug. 23, 1990; WO 92/07945 publishesMay 14, 1992 and in EP Patent publication 0 453 242 published Oct. 23,1991 relating the teachings of which we herein incorporates byreference. Exemplary vectors using poxvirus sequences are disclosed inEP Patent 0 110 385 published Jun. 13, 1984.

Yet another alternative is to incorporate VZV DNA sequences lackingORFS/L sequences into a non-VZV viral vector or genome, such as, herpessimplex virus. As used here, viral vector means a nucleic acid moleculein which a gene sequence to be transferred is fused to a subset of viralsequences that are capable of expressing the gene at some point in theviral lifecycle.

VZV DNA sequences lacking ORF/L sequences are inserted into non-VZVviral vectors or genomes using the cosmid techniques discussed herein toreconstruct VZV from four cosmids, with overlapping VZV DNA fragments.When a non-VZV viral vector or genome or chimeric genome is desired atleast one, or part of one, of the four fragments used in the cosmidtechnique to reconstruct a VZV virus will be a non-VZV, viral genomefragment.

C. Production of Mutant VZV

VZV with a mutant or wild type ORFS/Ls will be grown in tissue culturecells. For experiments with mammals, not including humans, cells such ashuman foreskin fibroblasts (HF), human neuroblastoma cells (SK-N-SH),human or murine neuroblastoma cells or cell lines, MRC-5 cells, VEROcells, or MeWO cells will be used to propagate the virus. Mutant VZVvirus will be harvested from cultures of these cells described. Theisolated mutant virus will then be further studied for its ability toelicit an immune response and/or provide protection against VZVinfection.

Mutant VZV for use in humans will be produced from an FDA approved cellline in large scale amounts. Such cells include MRC-5 or WI-38 cells(both are primary human diploid fibroblasts) or VERO cells. The mutantVZV will be generated in the production cell line either by transfectionusing overlapping VZV fragments from four cosmids as discussed below ortransfection of viral DNA or capsids prepared from mutant VZV isolatedfrom another cell line. Either method of transfection will prevent thecontamination of FDA approved cells with adventitious agents orcontaminants from a non-qualified cell line.

A mutant VZV produced from the above cell lines will be used to infectprogressively larger flasks of tissue culture cells. Infected cells willbe used as subsequent inoculums. Viable infected tissue culture cellsare removed from the tissue culture vessels using trypsin and added to a1 to 100 fold (or more) excess of uninfected cells to accomplishprogressively larger inoculations. Once an optimal yield is obtained themutuant VZV will be harvested from the tissue culture cells. Thisprocess can be repeated until a large scale production is achieved.Infected cells will be removed from the tissue culture vessel anddisrupted using for example, sonication, dounce homogenization or somecombination of the above. The viruses are then isolated from cellularmaterial using centrifugation techniques known in the art. Once thevirus is isolated a stabilizing agent is added, such as a carbohydrateor carbohydrate derivative and the virus is then aliquoted andlyophilized.

D. VZV Vaccines and Immunogenic Compositions

VZV vaccines or immunogenic compositions can be administered to subjectsto prevent VZV infections. Both primary and secondary infections can beprevented. The vaccine prevents varicella infections by stimulating theimmune system with an attenuated virus incapable of fully manifestingthe disease. The vaccine prevents zoster infections because changes inthe ORFS/L gene interfere with the virus' ability to reactivate.

The VZV vaccines described in this invention are administered to preventthe acquisition of chicken pox or shingles by inducing immunity insubjects not previously exposed to the virus in recipients of thevaccine or to subjects previously exposed to the virus in order toprevent zoster. The therapeutic administration of the vaccine shouldhelp to reduce the possibility of developing shingles in adult vaccinesdue to a VZV strain they acquired earlier than vaccination. The vaccinecan be used in mammals, preferably primates, such as monkey or gorilla,and most preferably in humans.

A major advantage of the VZV vaccine provided herein is that it will notreactivate to cause shingles. VZV(Oka) has been documented to reactivateand cause shingles in certain populations. Reactivation is also known tooccur in the native herpes simplex virus. However, in herpes simplexvirus lacking the γ₁ 34.5 gene linked to reactivation has beendemonstrably reduced in animal models. By deleting or modifying theORFS/L gene in VZV it is anticipated that a similar reduction inreactivation frequency will result. Two reading frames of the VZV ORFS/L(Dumas) gene, the first (VZV genomic sequence 1-562) and second (VZVgenomic sequence 105,125-105,013 appended to 105,012-104,925) sharesimilar positions to the two reading frames of herpes simplex virus γ₁34.5 gene in their respective genomes, although the ORFS/L gene and theγ₁ 34.5 gene are not considered structural homologs. In the VZV genome,corresponding parts of the ORFS/L gene are located just proximal to theU_(L) 5' repeat region and in the IR_(L) regions, while in the γ₁ 34.5gene is located at the U_(L) 3' and 5' repeat region of herpes simplexvirus-1. Because the γ₁ 34.5 gene has been linked to a reduction ofherpes simplex virus-1 reactivation in animal models, mutations to theORFS/L gene from VZV will generate a vaccine disabled in its ability toreactivate and cause shingles.

VZV(Oka) will serve as the parent strain due to its ability to stimulatean immune response and protect subjects from getting chicken pox. Thevaccines derived using a mutated ORFS/L gene from an Oka strain will beunable to cause shingles, unlike Oka. Different VZV strains, other thanOka, can be used with a disabled ORFS/L gene to generate an attenuatedvirus, such as: RIT/Oka, Merck/Oka, Ellen, EF, Webster A, Yamada, Izawa,Tsuchiyama, Watanabe, Wada, Terada, Kawaguchi, Inoue, SY, Scott, KMcC,AW, H-551, CP5,262, 80-2, 1294, 6050 and delta (Simian varicella virus).Clinical isolates with better growth or immune stimulation propertiesare preferred strains for making vaccines with the invention.

To make a VZV vaccine or immunogenic composition a modified ORFS/L genewill be produced in a VZV virus as discussed herein. The effectivenessof the vaccine in preventing Varicella infections will be measured inhumans. Humans will be first inoculated with PFU's ranging from100-20,000 PFU of mutant VZV per inoculation, PFUs are measured asdiscussed herein. After the first inoculation, a second boosterinjection of similar or increased dosage usually will be given. Subjectswill be exposed to wildtype VZV after the first or second inoculationand the occurrence of varicella infections observed. Potential sideeffects of the vaccine will be monitored in volunteer adults previouslyexposed to VZV, before inoculating subjects that have not ever developedvaricella infections. Attenuated virus is used without an adjuvant andwith a physiologically suitable carrier.

As is known in the art and discussed herein, the modified DNA, in thiscase ORFS/L DNA containing mutations in the sequence, is inserted intothe viral genome using, for example, homologous recombinationtechniques. The insertion is generally made into a gene which isnon-essential in nature, for example, the thymidine kinase gene (tk),which also provides a selectable marker. Plasmid shuttle vectors thatgreatly facilitate the construction of recombinant viruses have beendescribed (see, for example, Mackett, et al. (1984); Chakrabarti, et al.(1985); Moss (1987)). Expression of the heterologous polypeptide thenoccurs in cells or individuals which are immunized with the liverecombinant virus.

E. Diagnostic Assays and Use as a Vaccine Marker

The novel ORFS/L gene of the present invention can be used in diagnosticassays to detect VZV in a sample, to detect ORFS/L-like genes and todetect strain differences among ORFS/L genes using either chemicallysynthesized or recombinant ORFS/L gene fragment. Additionally, the novelORFS/L gene can be used as a vaccine marker to differentiate between anindividual or sample infected with or containing wild type VZV and anindividual or sample infected with or containing a VZV vaccine, ie., alive attenuated VZV vaccine currently in use such as the Oka vaccine. Inyet another embodiment, fragments of the ORFS/L gene can also be linkedto secondary nucleic acids with sequences that either bind a solidsupport or other detection probes for use in diagnostic assays.

In one aspect of the invention fragments of the ORFS/L gene comprisingat least between 10 and 20 nucleotides can be used as primers to amplifynucleic acids using polymerase chain reaction (PCR) methods well knownin the art and as probes in nucleic acid hybridization assays to detecttarget genetic material such as VZV DNA in clinical specimens (with orwithout PCR). See for example, U.S. Pat. Nos. 4,683,202; 4,683,195;5,091,310; 5,008,182 and 5,168,039. In an exemplary assay, a conservedregion of the ORFS/L gene among virus variants is selected, for examplenucleotides 70 through 800, as shown in FIG. 6a, as the sequence to beamplified and detected in the diagnostic assay. Oligonucleotide primersat least substantially complementary to (but preferably identical with)the sequence to be amplified are constructed and a sample suspected ofcontaining a VZV nucleic acid sequence to be detected is treated withprimers for each strand of VZV nucleic acid sequence to be detected,four different deoxynucleotide triphosphates and a polymerization agentunder appropriate hybridization conditions such that an extensionproduct of each primer is synthesized that is complementary to the VZVnucleic acid sequences suspected in the sample, which extension productssynthesized from one primer, when separated from its complement canserve as a template for synthesis of the extension product of the otherprimer in a polymerase chain reaction. After amplification, the productof the PCR can be detected by the addition of a labeled probe, likewiseconstructed from the ORFS/L sequence, capable of hybridizing with theamplified sequence as is well known in the art. See, e.g. U.S. Pat. No.5,008,182. In another embodiment the ORFS/L gene probes or primers canbe used in a vaccine marker assay to detect a vaccine or wild typeinfection. In this assay regions of the ORFS/L gene with the leasthomology between the vaccine and the wild type are preferred for use asprimers or probes. Alternatively, introduction of a restriction siteinto the ORFS/L gene will provide an ORFS/L gene vaccine marker that canbe used with PCR fragments to detect such differences in a restrictiondigest. Such procedures and techniques for detecting sequence variants,such as, point mutations with the expected location or configuration ofthe mutation, are known, are already known in the art and have beenapplied in the detection of sickle cell anemia, hemoglobin C disease,diabetes and other diseases and conditions as disclosed in U.S. Pat. No.5,137,806. These methods are readily applied by one skilled in the artto detect and differentiate between wild type and vaccine infections inVZV.

In another embodiment the ORFS/L gene can be used in its entirety or asfragments to detect the presence of ORFS/L gene, related genes, orORFS/L gene transcription products in cells, tissues, samples and thelike using hybridization probe techniques known in the art or inconjunction with one of the methods discussed herein. When used as ahybridization probe, fragments of the ORFS/L gene are preferably 50-200nucleotides long, whole preferably 100-300 nucleotides long and mostpreferably greater than 300 nucleotides long.

F. ORFS/L Vectors and Chimeric VZV Virus Production

The ORFS/L gene can be expressed in different vectors using differenttechniques known in the art resulting in the generation of chimeric VZVvirus. Useful and known techniques include marker transfer or homologousrecombination, direct in vitro ligation, defective vector technology andamplicon generation (see, e.g., Frenkel, N. et al., Gene Transfer andCancer, edited by M. L. Pearson and N. L. Sternberg (1984), Kwong, A. D.and Frenkel, Virology 142, 421-425(1985); U.S. patent (Ser. No.07/923,015 by Roizman). Vectors used in such techniques include cosmids,plasmids, and infective or defective viruses. Such vectors are known inthe art. (A cosmid as used herein is a plasmid containing a lambdabacteriophage cos site. The cos site is the cis signal for packaginglambda DNA. Therefore, a cosmid, unlike a plasmid, can be packaged withhigh efficiency into a lambda head in vitro. This technique allowscloning of very large (30-45 kbp) fragments of DNA.) The vectors can beeither single stranded or double stranded and made of either DNA or RNA.

Generally, the ORFS/L gene is inserted into the vector alone or linkedto other VZV genomic DNA. In direct in vitro ligation applications, theisolated ORFS/L gene alone is used. In homologous recombination andmarker transfer flanking nucleic acid sequences are required to effecttransfer of the ORFS/L sequence into a VZV viral genome. For ORFS/L geneuse in viral complementation using cosmids and other vectors discussedherein the ORFS/L gene (or a fragment of the gene) in a vector ispreferably operatively linked to at least 1 kb of VZV genomic nucleicacid and more preferably at least 5 kb of VZV nucleic acid. The VZVgenomic nucleic acid can be on one side or both sides of the ORFS/Lgene. If only a specific region of the ORFS/L gene is to be used togenerate a mutant VZV virus, an open reading frame or fragment of theORFS/L gene is inserted into a vector. Preferably, the ORFS/L gene,operatively linked to a vector, has a mutation.

G. ORFS/L Protein

Another aspect of the invention includes the isolated ORFS/L proteinencoded by the ORFS/L gene DNA sequence as taught herein. The ORFS/Lprotein can be used to study and modify the life cycle of VZV becausethe ORFS/L gene uniquely forms a complete gene during two time specificperiods in the VZV viral life cycle, during concatamer DNA formation andduring circular DNA formation. The novel VZV ORFS/L gene is alsoimplicated in cell apoptosis by virtue of its similarity with the γ₁34.5 gene of herpes simplex virus. This similarity makes ORFS/L proteina useful tool and therapeutic for studying and treating degenativediseases of the central nervous system caused by cell death.

The ORFS/L proteins comprise a Dumas protein with 224 amino acids(24,265 daltons) as shown in FIG. 5, an Oka protein with 223 amino acids(23,775 daltons) as shown in FIG. 6a and an Oka protein with 157 aminoacids (17,123 daltons) as shown in FIG. 6b. The term "ORFS/L protein,"refers to the sequences shown in FIGS. 5, 6a and 6b, and the sequencelisting, unless a specific strain is indicated. The term "ORFS/Lprotein" as used herein refers to an ORFS/L protein from any strain ofVZV and to proteins that are at least 90% homologous to the ORFS/L aminoacid sequences show in FIGS. 5 and 6. The ORFS/L protein can be modifiedto affect VZV life cycle or apoptosis by deletion, insertion andsubstitution into the ORFS/L gene DNA sequence, as discussed herein, orby chemical synthesis of different amino acid sequence or by chemicalmodification. Truncated ORFS/L proteins can be formed by deletion of aportion of the ORFS/L DNA sequence or the introduction of terminationsignal(s) into the ORFS/L gene DNA sequence. Preferred deletions to theORFS/L protein correspond to deleted amino acid sequence or sequencesthat contain at least one amino acid selected from the group consistingof Glu, Asp, Arg, Lys, Cys and Pro. More preferably at the deleted aminoacid sequence or sequences contain at least two amino acids selectedfrom the group consisting of Glu, Asp, Arg, Lys, Cys and Pro. Morepreferably the deleted amino acid sequence or sequences contain at leasttwo prolines. Also, preferred are mutations related to mutant VZVdiscussed herein, where ORFS/L protein mutations prevent shingles ordecrease viral propagation in cells of neuronal tissue origin.

Other mutations of the ORFS/L protein useful in modifying VZV life cycleor apoptopsis include, but are not limited to, modification of cAMPphosphorylation (Arg/Lys-Arg/Lys-X-X-Asp/Glu) and/or, myristylizationsites (Glycine-XI-X2-X2-Ser/Thr-X-X-Asp/Glu; where X1 is notGlu,Asp,Arg, Lys, His Pro, Phe, Tyr, Trp, where X2 is any amino acid andwhere X3 is not Pro), or modification of the PKC phosphorylation sites(Ser/Thr-X-Arg/Lys) and/or N-linked glycosylation sites (Asn-X-Ser/Thr;where X is not Pro).

The ORFS/L gene or fragments thereof can be expressed in a mammalian,insect, or microorganism host. The polynucleotide encoding ORFS/L genesare inserted into a suitable expression vector compatible with the typeof host cell employed and is operably linked to the control elementswithin that vector. Vector construction employs techniques which areknown in the art. Site-specific DNA cleavage involved in suchconstruction is performed by treating with suitable restriction enzymesunder conditions which generally are specified by the manufacturer ofthese commercially available enzymes. A suitable expression vector isone that is compatible with the desired function (e.g., transientexpression, long term expression, integration, replication,amplification) and in which the control elements are compatible with thehost cell.

Mammalian Cell Expression

Vectors suitable for replication in mammalian cells are known in theart, and can include viral replicons, or sequences that ensureintegration of the sequence encoding ORFS/L into the host genome.Exemplary vectors include those derived from simian virus SV40,retroviruses, bovine papilloma virus, vaccinia virus, and adenovirus.

Such suitable mammalian expression vectors contain a promoter to mediatetranscription of foreign DNA sequences and, optionally, an enhancer.Suitable promoters are known in the art and include viral promoters suchas those from SV40, cytomegalovirus (CMV), Rous sarcoma virus (RSV),adenovirus (ADV), and bovine papilloma virus (BPV).

The optional presence of an enhancer, combined with the promoterdescribed above, will typically increase expression levels. An enhanceris any regulatory DNA sequence that can stimulate transcription up to1000-fold when linked to endogenous or heterologous promoters, withsynthesis beginning at the normal mRNA start site. Enhancers are alsoactive when placed upstream or downstream from the transcriptioninitiation site, in either normal or flipped orientation, or at adistance of more than 1000 nucleotides from the promoter. See Maniatis,Science 236:1237(1987), Alberts, Molecular Biology of the Cell, 2nd Ed.(1989). Enhancers derived from viruses may be particularly useful,because they typically have a broader host range. Examples include theSV40 early gene enhancer (see Dijkema, EMBO J. 4:761(1985)) and theenhancer/promoters derived from the long terminal repeat (LTR) of theRSV (see Gorman, Proc. Natl. Acad. Sci. 79:6777(1982b)) and from humancytomegalovirus (see Boshart, Cell 41:521(1985)). Additionally, someenhancers are regulatable and become active only in the presence of aninducer, such as a hormone or metal ion (see Sassone-Corsi and Borelli,Trends Genet. 2:215(1986)); Maniatis, Science 236:1237(1987)). Inaddition, the expression vector can and will typically also include atermination sequence and poly(A) addition sequences which are operablylinked to the VZV ORFS/L coding sequence.

Sequences that cause amplification of the gene may also be desirablyincluded in the expression vector or in another vector that isco-translated with the expression vector containing an ORFS/L DNAsequence, as are sequences which encode selectable markers. Selectablemarkers for mammalian cells are known in the art, and include forexample, thymidine kinase, dihydrofolate reductase (together withmethotrexate as a DHFR amplifier), aminoglycoside phosphotransferase,hygromycin B phosphotransferase, asparagine synthetase, adenosinedeaminase, metallothionien, and antibiotic resistant genes such asneomycin.

The vector that encodes an ORFS/L protein or polypeptide can be used fortransformation of a suitable mammalian host cell. Transformation can beby any known method for introducing polynucleotide into a host cell,including, for example packaging the polynucleotide in a virus andtransducing a host cell with the virus. The transformation procedureused depends upon the host to be transformed. Methods for introductionof heterologous polynucleotide into mammalian cells are known in the artand include dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g.,Hep G2), and a number of other cell lines.

Insect Cell Expression

The components of an insect cell expression system include a transfervector, usually a bacterial plasmid, which contains both a fragment ofthe baculovirus genome, and a convenient restriction site for insertionof the heterologous gene or genes to be expressed; a wild typebaculovirus with a sequence homologous to the baculovirus-specificfragment in the transfer vector (this allows for the homologousrecombination of the heterologous gene in to the baculovirus genome);and appropriate insect host cells and growth media. Exemplary transfervectors for introducing foreign genes into insect cells include pAc373and pVL985. See Luckow and Summers, Virology 17:31(1989).

The plasmid can also contains the polyhedron polyadenylation signal anda procaryotic ampicillin-resistance (amp) gene and origin of replicationfor selection and propagation in E. coli. See Miller, Ann. Rev.Microbiol. 42:177(1988).

Baculovirus transfer vectors usually contain a baculovirus promoter,i.e., a DNA sequence capable of binding a baculovirus RNA polymerase andinitiating the downstream (5' to 3') transcription of a coding sequence(e.g., structural gene) into mRNA. The promoter will have atranscription initiation region which is usually placed proximal to the5' end of the coding sequence and typically includes an RNA polymerasebinding site and a transcription initiation site. A baculovirus transfervector can also have an enhancer, which, if present, is usually distalto the structural gene. Expression can be either regulated orconstitutive.

Yeast and Bacteria Expression

A yeast expression system can typically include one or more of thefollowing: a promoter sequence, fusion partner sequence, leadersequence, transcription termination sequence. A yeast promoter, capableof binding yeast RNA polymerase and initiating the downstream (3')transcription of a coding sequence (e.g. structural gene) into mRNA,will have a transcription initiation region usually placed proximal tothe 5' end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site (a "TATA Box") and atranscription initiation site. The yeast promoter can also have anupstream activator sequence, usually distal to the structural gene. Theactivator sequence permits inducible expression of the desiredheterologous DNA sequence. Constitutive expression occurs in the absenceof an activator sequence. Regulated expression can be either positive ornegative, thereby either enhancing or reducing transcription.

Particularly useful yeast promoters include alcohol dehydrogenase (ADH)(EP Patent Pub. No. 284 044), enolase, glucokinase, glucose-6-phosphateisomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH),hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvatekinase (PyK) (EP Patent Pub. No. 329 203). The yeast PHO5 gene, encodingacid phosphatase, also provides useful promoter sequences. SeeMyanohara, Proc. Natl. Acad. Sci. USA 80:1(1983).

An ORFS/L gene or an active fragment thereof can be expressedintracellularly in yeast. A promoter sequence can be directly linkedwith an ORFS/L gene or fragment, in which case the first amino acid atthe N-terminus of the recombinant protein will always be a methionine,which is encoded by the ATG start codon. If desired, methionine at theN-terminus can be cleaved from the protein by in vitro incubation withcyanogen bromide.

Intracellularly expressed fusion proteins provide an alternative todirect expression of an ORFS/L sequence. Typically, a DNA sequenceencoding the N-terminal portion of a stable protein, a fusion partner,is fused to the 5' end of heterologous DNA encoding the desiredpolypeptide. Upon expression, this construct will provide a fusion ofthe two amino acid sequences. For example, the yeast or human superoxidedismutase (SOD) gene, can be linked at the 5' terminus of an ORFS/Lsequence and expressed in yeast. The DNA sequence at the junction of thetwo amino acid sequences may or may not encode a clearable site. See,e.g., EP Patent Pub. No. 196 056. Alternatively, ORFS/L polypeptides canalso be secreted from the cell into the growth media by creating afusion protein comprised of a leader sequence fragment that provides forsecretion in yeast or bacteria of the ORFS/L polypeptides. Preferably,there are processing sites encoded between the leader fragment and theORFS/L sequence that can be cleaved either in vivo or in vitro. Theleader sequence fragment typically encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell. DNA encoding suitable signal sequences can be derived fromgenes for secreted yeast proteins, such as the yeast invertase gene (EPPatent Pub. No. 12 873) and the A-factor gene (U.S. Pat. No. 4,588,684).Alternatively, leaders of non-yeast origin, such as an interferonleader, can be used to provide for secretion in yeast (EP Patent Pub.No. 60057). Transcription termination sequences recognized by yeast areregulatory regions located 3' to the translation stop codon. Togetherwith the promoter they flank the desired heterologous coding sequence.These flanking sequences direct the transcription of an mRNA which canbe translated into the ORFS/L polypeptide encoded by the ORFS/L DNA.

Typically, the above described components, comprising a promoter, leader(if desired), coding sequence of interest, and transcription terminationsequence, are put together in plasmids capable of stable maintenance ina host, such as yeast or bacteria. The plasmid can have two replicationsystems, so it can be maintained as a shuttle vector, for example, inyeast for expression and in a procaryotic host for cloning andamplification. Examples of such yeast-bacteria shuttle vectors includeYEp24 (see Botstein, Gene 8:17-24 (1979)), pC1/1 (see Brake, Proc. Natl.Acad. Sci. USA 81:4642-4646(1984)), and YRp17 (see Stinchcomb, J. Mol.Biol. 158:157(1982)). In addition, the plasmid can be either a high orlow copy number plasmid. A high copy number plasmid will generally havea copy number ranging from about 5 to about 200, and typically about 10to about 150. A host containing a high copy number plasmid willpreferably have at least about 10, and more preferably at least about20. Either a high or low copy number vector may be selected, dependingupon the effect on the host of the vector and the ORFS/L polypeptides.See, e.g., Brake, et al., supra.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors typicallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. See Orr-Weaver, Methods InEnzymol. 101:228-245(1983) and Rine, Proc. Natl. Acad. Sci. USA80:6750(1983).

Typically, extrachromosomal and integrating expression vectors cancontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers can include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP1, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker can also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP1 allows yeast to grow in the presence of copper ions.See Butt, Microbiol. Rev. 51:351(1987).

Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors aretypically comprised of a selectable marker that is either maintained ina replicon or developed into an integrating vector, as described above.Expression and transformation vectors, either extrachromosomal orintegrating, have been developed for transformation into many yeasts.Exemplary yeasts cell lines are Candida albicans (Kurtz, Mol. Cell.Biol. 6:142(1986), Candida maltosa (Kunze, J. Basic Microbiol.25:141(1985), Hansenula polymorpha (Gleeson, J. Gen. Microbiol.132:3459(1986) and Roggenkamp, Mol. Gen. Genet. 202:302(1986),Kluyveromyces fragilis (Das, J. Bacteriol. 158:1165(1984), Kluyveromyceslactis (De Louvencourt, J. Bacteriol. 154:737(1983) and Van den Berg,Bio/Technology 8:135(1990), Pichia guillerimondii (Kunze, J. BasicMicrobiol. 25:141(1985), Pichia pastoris (Cregg, Mol. Cell. Biol. 5:3376(1985), Saccharomyces cerevisiae (Hinnen, Proc. Natl. Acad. Sci. USA75:1929(1978) and Ito, J. Bacteriol. 153:163(1983), Schizosaccharomycespombe (Beach and Nurse, Nature 300:706(1981), and Yarrowia lipolytica(Davidow, Curr. Genet. 10:380471(1985) and Gaillardin, Curr. Genet.10:49(1985).

Methods of introducing exogenous DNA into yeast hosts are well-known inthe art, and typically include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed. Seethe publications listed in the foregoing paragraph for appropriatetransformation techniques.

Additionally, the ORFS/L gene or fragment thereof can be expressed in abacterial system. In such system, a bacterial promoter is any DNAsequence capable of binding bacterial RNA polymerase and initiating thedownstream (3') transcription of a coding sequence (e.g. a desiredheterologous gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5' end of thecoding sequence. This transcription initiation region typically includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter can also have a second domain called an operator,that can overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein can bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression can occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation can be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5') to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in Escherichia coli(E. coli). See Raibaud, Ann. Rev. Genet. 18:173(1984). Regulatedexpression can therefore be either positive or negative, thereby eitherenhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) (see Chang,Nature 198:1056(1977), and maltose. Additional examples include promotersequences derived from biosynthetic enzymes such as tryptophan (trp)(see Goeddel, NUC. ACIDS RES. 8:4057(1981), Yelverton, Nuc. Acids Res.9:731(1981), U.S. Pat. No. 4,738,921 and EP Patent Pub. Nos. 36 776 and121 775). The lactomase (bla) promoter system (see Weissmann, Interferon3 (ed. I. Gresser), the bacteriophage lambda PL promoter system (seeShimatake, Nature 292:128(128) and the T5 promoter system (U.S. Pat. No.4,689,406) also provides useful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter can be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter such as the tac promoter (see U.S.Pat. No. 4,551,433, Amann, Gene 25:167(1983) and de Boer, Proc. Natl.Acad. Sci. 80:21(1983)). A bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is exemplary. (see Studier, J. Mol. Biol. 189:113(1986) andTabor, Proc. Natl. Acad. Sci. 82:1074(1985)).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of the ORFS/L gene orfragment thereof in prokaryotes. In E. coli, the ribosome binding siteis called the Shine-Dalgarno (SD) sequence and includes an initiationcodon (ATG) and a sequence 3-9 nucleotides in length located 3-11nucleotides upstream of the initiation codon (see Shine, Nature254:34(1975). The SD sequence is thought to promote binding of mRNA tothe ribosome by the pairing of bases between the SD sequence and the 3'and of E. coli 16S rRNA (see Steitz, Biological Regulation andDevelopment: Gene Expression (ed. R. F. Goldberger)(1979)).

ORFS/L protein can be expressed intracellularly. A promoter sequence canbe directly linked with an ORFS/L gene or a fragment thereof, in whichcase the first amino acid at the N-terminus will always be a methionine,which is encoded by the ATG start codon. If desired, methionine at theN-terminus can be cleaved from the protein by in vitro incubation withcyanogen bromide or by either in vivo on in vitro incubation with abacterial methionine N-terminal peptidase. See EP Patent Pub. No. 219237.

Fusion proteins provide an alternative to direct expression. Typically,a DNA sequence encoding the N-terminal portion of an endogenousbacterial protein, or other stable protein, is fused to the 5' end ofheterologous ORFS/L gene coding sequences. Upon expression, thisconstruct will provide a fusion of the two amino acid sequences. Forexample, the bacteriophage lambda cell gene can be linked at the 5'terminus of an ORFS/L gene or fragment thereof and expressed inbacteria. The resulting fusion protein preferably retains a site for aprocessing enzyme (factor Xa) to cleave the bacteriophage protein fromthe ORFS/L gene or fragment thereof (see Nagai, Nature 309:810(1984).Fusion proteins can also be made with sequences from the lacZ gene (Jia,Gene 60:197(1987), the trpE gene (Allen, J. Biotechnol. 5:93(19857) andMakoff, J. Gen. Microbiol. 135:11(1989), and the Chey gene (EP PatentPub. No. 324 647) genes. The DNA sequence at the junction of the twoamino acid sequences may or may not encode a clearable site. Anotherexample is a ubiquitin fusion protein. Such a fusion protein is madewith the ubiquitin region that preferably retains a site for aprocessing enzyme (e.g., ubiquitin specific processing-protease) tocleave the ubiquitin from the ORFS/L polypeptide. Through this method,mature ORFS/L polypeptides can be isolated. See Miller, Bio/Technology7:698(1989).

Alternatively, ORFS/L proteins or polypeptides can also be secreted fromthe cell by creating chimeric DNA molecules that encode a fusion proteincomprised of a signal peptide sequence fragment that provides forsecretion of the ORFS/L proteins or polypeptides in bacteria. (See, forexample, U.S. Pat. No. 4,336,336). The signal sequence fragmenttypically encodes a signal peptide comprised of hydrophobic amino acidswhich direct the secretion of the protein from the cell. The protein iseither secreted into the growth media (gram-positive bacteria) or intothe periplasmic space, located between the inner and outer membrane ofthe cell (gram-negative bacteria). Preferably there are processingsites, which can be cleaved either in vivo or in vitro encoded betweenthe signal peptide fragment and the ORFS/L protein or polypeptide.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) (Masui, Experimental Manipulation of Gene Expression (1983)and Ghrayeb, EMBO J. 3:2437(1984)) and the E. coli alkaline phosphatasesignal sequence (phoA) (see Oka, Proc. Natl. Acad. Sci. 82:7212(1985).The signal sequence of the alpha-amylase gene from various Bacilusstrains can be used to secrete heterologous proteins from B. subtilis(see Palva, Proc. Natl. Acad. Sci. 79:5582(1982) and EP Patent Pub. No.244 042).

Transcription termination sequences recognized by bacteria areregulatory regions located 3' to the translation stop codon. Togetherwith the promoter they flank the coding sequence. These sequences directthe transcription of an mRNA which can be translated into the ORFS/Lprotein or polypeptide encoded by the ORFS/L DNA sequence. Transcriptiontermination sequences frequently include DNA sequences of about 50nucleotides capable of forming stem loop structures that aid interminating transcription. Examples include transcription terminationsequences derived from genes with strong promoters, such as the t genein E. coli as well as other biosynthetic genes.

Typically, the promoter, signal sequence (if desired), coding sequenceof interest, and transcription termination sequence are maintained in anextrachromosomal element (e.g., a plasmid) capable of stable maintenancein the bacterial host. The plasmid will have a replication system, thusallowing it to be maintained in the bacterial host either for expressionor for cloning and amplification. In addition, the plasmid can be eithera high or low copy number plasmid. A high copy number plasmid willgenerally have a copy number ranging from about 5 to about 200, andtypically about 10 to about 150. A host containing a high copy numberplasmid will preferably contain at least about 10, and more preferablyat least about 20 plasmids.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectorstypically contain at least one sequence homologous to the bacterialchromosome that allows the vector to integrate. Integrations appear toresult from recombinations between homologous DNA in the vector and thebacterial chromosome. See e.g., EP Patent Pub. No. 127 328.

Typically, extrachromosomal and integrating expression constructs cancontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and can include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline (see Davies, Ann. Rev. Microbiol.32:469(1978). Selectable markers can also include biosynthetic genes,such as those in the histidine, tryptophan, and leucine biosyntheticpathways.

Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are typicallycomprised of a selectable marker that is either maintained in anextrachromosal vector or an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal orintegrating, have been developed for transformation into many bacteria.Exemplary are the expression vectors disclosed in Palva, Proc. Natl.Acad. Sci. 79:5582(1982), EP Patent Pub. Nos. 036 259 and 063 953 andPCT Patent Publication WO 84/04541 (for B. subtilis); in Shimatake,Nature 292:128(1981), Amann, Gene 40:183(1985), Studier, J. Mol. Biol.189:113(1986) and EP Patent Pub. Nos. 036 776, 136 829 and 136 907 (forE. coli); in Powell, Appl. Environ. Microbiol. 54:655(1988) and U.S.Pat. No. 4,745,056 (for Streptococcus).

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and typically include either the transformation of bacteriatreated with CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Exemplary methodologies can be found in Masson, FEMS Microbiol. Let.60:273(1989), Palva, Proc. Natl. Acad. Sci. 79:5582(1982), EP PatentPub. Nos. 036 259 and 063 953 and PCT Patent Pub. WO 84/04541 forBacillus transformation. For campylobacter transformation, see e.g.,Miller, Proc. Natl. Acad. Sci. 85:856(1988) and Wang, J. Bacteriol.172:949(1990). For E. coli, see e.g., Cohen, Proc. Natl. Acad. Sci.69:2110(1973), Dower, Nuc. Acids Res. 16:6127(1988), Kushner, GeneticEngineering: Proceedings of the International Symposium on GeneticEngineering (eds. H. W. Boyer and S. Nicosia), Mandel, J. Mol. Biol.53:159(1970) and Taketo, Biochem. Biophys. Acta 949:318(1988). ForLactobacillus and Pseudomonas, see e.g., Chassy, FEMS Microbiol. Let.44:173(1987) and Fiedler, Anal. Biochem. 170:38(1988), respectively. ForStreptococcus, see e.g., Augustin, FEMS Microbiol. Let. 66:203(1990),Barany, J. Bacteriol. 144:698(1980), Harlander, Streptococcal Genetics(ed. J. Ferretti and R. Curtiss III)(1987), Perry, Infec. Immun.32:1295(1981), Powell, Appl. Environ. Microbiol. 54:655(1988) andSomkuti, Proc. 4th Evr. Cong. Biotechnology 1:412(1987).

The present invention is illustrated by the following examples.

EXAMPLE 1

Identification of ORFS/L Gene in the Dumas Strain

The full DNA sequence of the Dumas strain of VZV is known. The linearDNA sequence of VZV (GenBank Accession Number X04370) was searched forsequences similar to the herpes simplex Virus γ₁ 34.5 gene. Computerassisted sequence analysis of VZV open reading frames (ORFs) yielded nosignificant homology to the γ₁ 34.5 gene.

The Dumas VZV genomic nucleotide sequence was further analyzed with theassistance of a computer. It was observed that out of the nine possiblereading frame orientations, a first ORF in the TR_(L) and U_(L) sequenceand a second ORF in the IR_(s) and IR_(L) sequence or a first ORF in theTR_(L) and U_(L) sequence and a third ORF in the TR_(S) sequence couldbe joined to produce a single reading frame of 224 amino acids. As shownin FIG. 2, these ORFs correspond to the joining of the striped regionrepresenting the first ORF in FIG. 2 with either of the 3' solid regionsrepresenting the second and third ORFs of the U_(L) -Par isomer shown.In the VZV lifecycle the ORFS/L gene is formed by different VZV genomicstructures, the U_(L) -Inv isomer, U_(L) -Par concatamer junctions orU_(L) -Par circles as shown in FIG. 2.

The ORFS/L (Dumas) gene shows no significant sequence homology to anygene using the standard PAM 120 alignment matrices for databasesearching. Using the BLOSUM62 alignment matrix, however, the herpessimplex γ₁ 34.5 gene and the ORFS/L (Dumas) gene scored a weak homology.(The databases searched were the Gen Bank, Swis-Prot, PDB, and PIRdatabases.) Because the proline content of both genes is high thishomology may be biased.

The full DNA sequence of the ORFS/L (Dumas) gene comprising the firstORFS/L reading frame joined to the second ORFS/L reading frame is shownin FIG. 5 along with its encoded amino acid sequence. The nucleic acidsequence of the ORFS/L (Dumas) gene comprised of the first ORFS/Lreading frame joined to the third ORFS/L reading frame is identical tothe ORFS/L (Dumas) gene made of the first and second reading frames.

The first ORF corresponds to the VZV (Dumas) genomic nucleotidesequences 1 through 562, including a stop codon ending at position 562.This nucleotide sequence encodes the C-terminal 168 amino acids of theORFS/L protein. The second ORF corresponds to the VZV (Dumas) genomicnucleotides 104,925 through 105,125 and the third ORF corresponds to theVZV (Dumas) genomic nucleotides 124,772 through 124,884. Either thesecond or third ORFs can encode the N-terminal 66 amino acids of theORFS/L protein. The ORFS/L (Dumas) gene nucleotide sequence andcorresponding 224 amino acid sequence is shown in FIG. 5 (SEQ ID NO:9and 10, respectively). A comparison of the Dumas ORFS/L gene nucleotidesequence with the PCR and genomic ORFS/L gene nucleotide sequences,discussed herein, is shown in FIG. 7.

ORFS/L gene has been refractory to discovery and is not listed as VZVopen reading frame with a translatable product in any of the known databanks, e.g., in GenBank. The standard linear display format used torepresent VZV sequences prevents the detection of a complete openreading frame. ORFS/L (Dumas) also lacks an adjacent canonical promoterTATA element and poly A addition signal AATAAA that are commonly used asgene identification criteria.

Although amino acid alignment revealed little or no homology betweenORFS/L and γ₁ 34.5, the position of ORFS/L gene in the VZV genome showssimilarity to the position of γ₁ 34.5 in the HSV genome. The γ₁ 34.5gene is located within the 8 kbp internal and terminal long repeatsequences in the herpes simplex virus genome. Its N-terminus is locatedin close proximity to the junction of the long and short regions and thegene is positioned immediately upstream of the potent transactivatorgene, ICP0. In similar manner, the N-terminal region of ORFS/L islocated near the long and short junction in VZV. By using the long-shortjunction as a point of reference, ORFS/L lies just upstream (but on theopposite end of the U_(L) segment) of the positional homolog to theherpes simplex virus ICP0 gene.

EXAMPLE 2

Identification and Isolation of ORFS/L (Oka) Gene in the Oka Strain

The existence of the ORFS/L gene in the Oka strain of VZV was confirmedand the DNA encoding the ORFS/L gene was isolated using a polymerasechain reaction (PCR)-based strategy using oligonucleotide primersdesigned from the sequence of VZV (Dumas) to amplify the DNA fragmentcontaining a complete ORFS/L (Oka) gene as shown in FIG. 3. Convenientrestriction enzyme sites were included at the 5' ends of the primers.Because linear molecules of VZV DNA (which comprise ˜95% of the packagedmolecules in VZV nucleocapsids) do not contain an intact ORFS/L sequencethey were not usable for the PCR cloning. Only genomes in which theU_(L) sequence has inverted (˜5% of packaged genomes) contain an intactORFS/L gene and are usable as shown in FIG. 3. Similarly, circular orconcatameric DNA also contain an intact version of the ORFS/L gene andwere amplifiable using PCR. These latter forms can be found during thereplication cycle of VZV.

PCR primers were generated based upon the sequence of VZV (Dumas).Primer 3 had the following sequence:

5'-gccgccaTGGAGGGGAGCGACGGAACACG-3' SEQ ID NO:1

The nucleotides in upper case correspond to nucleotides 124,326 to124,327 and the complementary strand of nucleotides 105,550 to 105,571of the VZV (Dumas) DNA sequence.

Primer 4 had the following sequence:

5'-gccgccatGGCTGTCGGCGGACTATGAAC-3' SEQ ID NO:2

The nucleotides in upper case correspond to the complementary strand ofnucleotides 1906 to 1926 of the VZV (Dumas) DNA sequence. In bothprimers, the lower case nucleotides add an Ncol site to the PCR productand do not correspond to VZV DNA sequence.

Primer 3 was designed to hybridize to viral DNA approximately 450nucleotides upstream from the predicted ORFS/L start codon and Primer 4was designed to hybridize to viral DNA approximately 60 bp downstream ofthe stop codon in the ORF2, leading to a predicted PCR productapproximately 2.5 kb in length as shown in FIG. 3.

PCR was performed using Primers 3 and 4 on viral DNA isolated fromeither the cytoplasm or nucleus of a VZV (Oka) infected human foreskinfibroblast culture using standard techniques. See, PCT application WO93/24616. A band having the predicted length was seen on an agarose gelafter the PCR reaction. The PCR reactions were treated with NcoI,fractionated on an agarose gel, and the proper sized DNA fragment wasisolated and ligated into the NcoI digested plasmid pGEM5Zf+. One clonefrom PCR material generated from the cytoplasmic DNA fraction wasisolated and designated pS/L C3.

EXAMPLE 3

Sequencing of the ORFS/L (Oka) Gene from a PCR Fragment

The SacI to BamHI fragment comprising the ORFS/L gene in the plasmidpS/L C3 of Example 2 above was isolated and inserted into the M13cloning vector, M13mp18. Specifically, a plaque containing theappropriate insert was isolated and designated M13S/LC3.

Primers were designed to hybridize to the S/L region of the M13 cloneand used to sequence the DNA of ORFS/L gene using the dideoxy chaintermination method of DNA sequencing. The resulting nucleotide sequenceand amino acid sequence is set forth in FIG. 6a.

EXAMPLE 4

Comparison of Oka ORFS/L Gene Sequence and the Dumas ORFS/L GeneSequence

The M13S/L C3 Oka ORFS/L gene sequence is 1121 nucleotides in length andcorresponds to the nucleotide sequence shown in FIG. 6a (SEQ ID NO:11).The M13S/L C3 Oka ORFS/L protein sequence is 223 amino acids in lengthand is shown in FIG. 6a (SEQ ID NO:12).

The entire ORF for M13S/L C3 aligns with the ORFS/L (Dumas) proteinsequence with a matching percentage of 84%.

In accordance with known techniques, all or a fragment of the nucleotidesequence embodying ORFS/L protein can be placed in an expression vectorsystem to produce all or a fragment of the ORFS/L protein orpolypeptide, as discussed previously. The ORFS/L protein can be purifiedusing standard techniques. The immunogenicity of the protein can beassessed using such techniques as are also known in the art. Forexample, guinea pigs can be inoculated intramuscularly with an admixtureof the purified, recombinantly produced, ORFS/L protein or fragmentthereof and complete Freund's adjuvant, followed by booster inoculationsone month later as is disclosed in U.S. Pat. No. 4,686,101 (hereinincorporated by reference in its entirety). Sera is then obtained fromthe guinea pigs and tested in an in vitro VZV neutralization assay asdescribed in Keller, J., Virology, 52:293 (1984) to determine whether itelicits VZV neutralizing antibodies in comparison to pre-immunizationsera.

EXAMPLE 5

Sequencing of the ORFS/L (Oka) Gene from VZV Genomic DNA

The ORFS/L gene of Oka was also sequenced using genomic VZV DNA. Asdiscussed herein VZV (Oka) DNA was cut into four overlapping fragmentsand placed into cosmids for amplication of VZV DNA for various purposes.Three plasmid, pV4L, pV21J and pV21S, were made from the cosmidsdescribed herein, and contain the first, second and third ORFs from theORFS/L gene. The plasmid names refer to the lefthand most region of theVZV genome (pV4L), the junction between IR_(L) and IR_(S) sequences inthe VZV genome (pV21J), and the righthand most region of the VZV genome(pV21S).

To produce pV4L the cosmid pVFsp4, described herein, was digested withBamHI to liberate a 920 base pair Oka DNA fragment (corresponding to thenucleotides 1 to 920 of the Dumas sequence). The 920 base pair fragmentwas isolated and inserted into BamHI digested pGEM7zf+ using standardtechniques. Restriction digests yielded the expected restrictionfragmentation patterns.

To produce pV21S, the cosmid pVSpe21, described herein, was digestedwith BamHI, to liberate a 1921 base pair Oka DNA fragment (correspondingto nucleotides 122,963 through 124,884 of the Dumas sequence). The 1921base pair fragment was isolated and inserted into BamHI digestedpGEM7zf+ using standard. Restriction digests of plasmid pV21S yieldedthe expected restriction fragmentation patterns.

To produce pV21J, the cosmid pVSpe21, described herein, was digestedwith BamHI and BglII to liberate a 2861 base pair Oka DNA fragment(corresponding to nucleotides 106,931 through 104,170 of the Dumasnucleotide sequence). The 2861 base pair fragment was isolated andinserted into a BamHI digested pGEM7zf+. Restriction digest of theplasmid pV21J yielded the expected restriction fragmentation patterns.

These three plasmids, pV4L, pV21J, and pV21S encoding the first, secondand third ORFs, respectively, were sequenced using double strandedsequencing techniques, as is well known in the art.

The nucleotide and amino acid sequences for the first ORF of the ORFS/Lgene of Oka are shown in FIG. 6b (SEQ ID NO:13 and 14, respectively).The nucleotide sequences of the second and third ORFs are nearlyidentical as shown in FIG. 7 (SEQ ID NO:15 and 16, respectively).

With respect to the PCR clone, p S/L C3, the nucleotide sequence of thefirst, second and third ORFs of Oka correspond to the respectivenucleotide sequences from p S/L C3, with the exception that there is aextra G in the first ORF of the Oka genomic DNA strain. The extra G inthe Oka strain is located in the corresponding location the Dumassequence between nucleotides 124,884 and 1 of the Dumas genomic sequence(the extra "G" arises from the unpaired 3' extension of the VZV genome).The extra G shifts the reading frame in Oka to make the start ATGcorrespond to nucleotide 88 of the Dumas genomic sequence. Instead ofencoding a protein identical to the 223 amino acid ORFS/L protein of thep S/L C3 DNA, the ORFS/L gene of Oka produces an 157 amino acid proteinwith a molecular weight of 17,123 daltons. The extra "G" is also shownand compared to the sequences described herein in FIG. 7.

EXAMPLE 6

Construction of Cosmid Clones of the VZV (Oka) Genome

Because VZV is difficult to grow in cell culture (see for example PCTpatent publication WO 93/24616 published Dec. 9, 1993), an alternativemethod making reconstructed VZV was developed. Briefly, four overlappingfragments spanning the entire VZV (Oka) genome were constructed,amplified and transfected into eukaryotic cells using four cosmids. Thefour cosmids are referenced as pV Fsp4, pV Spe5, pV Pme19, and pV Spe21and shown in FIG. 4. The overlapping VZV fragments in these cosmidsrecombine in the transfected cell, producing an intact reconstructedviral genome and an infectious reconstructed VZV. At least one of thefour cosmids can contain the desired mutation to the ORFS/L gene, aswell as, the viral genomic DNA. The general procedures set forth herecould be used for any strain of VZV or could be used to make chimericviruses and chimeric genomes with other viruses, such as herpes simplexvirus.

A. VZV DNA Isolation

To make these VZV cosmid clones, DNA was isolated from the nucleocapsidsof VZV (Oka) by the method of Straus (J. Virol 40:516-525, 1981), asfollows. Twelve 850 cm² roller bottles of confluent human foreskinfibroblast (HF) cells were infected with a frozen stock of previously,VZV (Oka) infected HF. Three days post infection (at a time when nearlyall the cells were rounded and showed cytopathic effect (CPE)) thenucleopcapsids were harvested.

The cells were shaken off the roller bottles into the media. The mediawas collected and centrifuged at approximately 200×g to pellet thecells. The cells were disrupted by freezing and thawing three timesalternately in a dry ice-acetone bath and 37° C. water bath. The cellswere resuspended in lysis buffer (30 mM Tris, pH 7.5; 0.5% NP-40; 0.5%deoxycholate; 3.6 mM CaCl₂ ; 5 mM Mg acetate; 125 mM KCl) containing 25μg/ml DNAseI and 25 μg/ml RNAseA. Following digestion for 30 minutes, at30° C., the lysate was extracted one time with 1/2 volume oftrichlorotrifluoroethane. The supernatant was placed on top of a stepgradient of 5% glycerol/40% glycerol in lysis buffer lacking CaCl₂ andmagnesium acetate. The material was centrifuged for 60 min. at 100,000×gat 4° C. The resulting pellet contained the viral nucleocapsids fromwhich VZV DNA was then isolated.

The nucleocapsid pellet was resuspended in 50 mM Tris, pH 7.5, 10 mMEDTA. SDS was added to a final concentration of 1% and proteinase K wasadded to a final concentration of 200 μg/ml. The nucleocapsids weredigested for 60 min at 65° C. The DNA was obtained by extracting thedigest three times with equal volumes of water saturated, bufferedphenol and chloroform (24 parts chloroform:1 part isoamyl alcohol). TheDNA was then dialyzed overnight against 10 mM Tris, pH 7.5/2 mM EDTA.

This DNA was the source of the DNA used for generating the cosmid clonesused in regenerating infectious mutant VZV virus as set forth herein.

B. Construction of Adaptors

In order to easily insert and excise VZV DNA into and from theconstructed cosmids, adaptors, short double-stranded oligonucleotideswith different termini, were synthesized on an Applied Biosystems(Foster City, Calif.) instrument according to manufacturer instructions.These adaptors were ligated to restriction digested VZV(Oka) DNA toyield restriction fragments compatible with the cosmid vector describedbelow. Three characteristics were incorporated into the design of theadaptors: 1. One end is blunt. This terminus was used to ligate to therestricted VZV DNA. 2. The opposing end of the adaptor was compatiblewith BamHI restricted DNA because the cosmid vector was to be digestedwith BamHI. 3. The internal nucleotides constituted an Asc1 restrictionenzyme site. Digestion of cosmids containing the restriction fragmentedVZV DNA ligated by these flanking adaptors separated the VZV DNA fromthe cosmid vector (because there are no AscI sites in the VZV(Oka)genome). The adapters used were:

    ______________________________________                                        Oligonucleotide: "Bam-Asc Adaptor"                                                                   "Blunt Asc"                                                          5'-GATCCGGCGCGCCA-3'                    5'-TGGCGCGCCG-3'                                            SEQ ID NO: 3                                                         SEQ ID NO: 4                                       ______________________________________                                    

Approximately 5,000 pmols of "Blunt Asc" was phosphorylated at the 5'end by incubation with ATP and T4 polynucleotide kinase.

Approximately 5,000 pmols of "Blunt Asc" and "Bam-Asc Adaptor" wereelectrophoresed onto a 20% polyacrylamide/7M urea gel. Followingelectrophoresis, the predominant bands were visualized by UV shadowingand excised from the surrounding gel. The oligonucleotides were elutedfrom the gel and purified on a Sep-pak C18 column according to themanufacturer's instructions. The quantity of each oligonucleotide wasassessed by spectrophotometry.

500 pmols each of "Bam-Asc Adaptor" and "Blunt Asc" were mixed together.The mixture was diluted to a final concentration of 20 pmol/μl of eacholigonucleotide; 50 mM NaCl; 10 mM Tris; 10 mM MgCl₂ ; 1 mM DTT, pH 7.9@ 25° C. The mixture was heated to 95° C. for 3 minutes and slowlycooled to room temperature (approximately 25° C.). This procedureproduced a double stranded oligo with the following characteristics:

5'-PO₄ -TGGCGCGCCG-3' SEQ ID NO: 4

3'-ACCGCGCGGCCTAG-OH-5' SEQ ID NO: 3

C. Ligation of Adaptors to VSV (Oka) DNA

The VZV DNA prepared as described in part A above was subjected torestriction digestion in order to generate four fragments for ligationinto four cosmids using the adapters. Based on the VZV genomic sequenceof the Dumas strain, the following four restriction fragments werepredicted to result from individual restriction digests using Fsp1, Spe1and Pme1 restriction enzymes.

Fsp1: nucleotides (L terminus) 1 to 33211

Spe1: nucleotides: 21875 to 62008

Pme1: nucleotides: 53877 to 96188

Spe1: nucleotides: 94208 to 124,884 (S terminus)

Restriction digests of VZV (Oka) DNA are consistent with these predictedsizes.

To generate these restriction fragments, approximately 2.5 μg of the VZV(Oka) nucleocapsid DNA form Part A above was separately digested witheither Fsp1, Spe1, or Pme1, as recommended by the manufacturer. The Spe1and Fsp1 digests were subsequently made blunt ended by incubation withdNTPs and T4 DNA polymerase. Pme1 releases the fragment as blunt endedand requires no such treatment.

100 pmols of the adaptors prepared in Part B above were then ligated tothe blunt ended VZV DNA. See Maniatis, T. et al, Molecular Cloning(1991). Following ligation, excess adaptors that had not ligated to theVZV DNA were removed by chromatography on a 2 ml Sepharose CL-4B columnequilibrated in 10 mM Tris, pH 7.5; 2 mM EDTA; 100 mM NaCl.

SuperCos™ 1 is a cosmid vector available from Stratagene (La Jolla,Calif.). The vector was prepared according to the manufacturer'sinstructions. In brief, the vector was digested with Xbal anddephosphorylated. The vector was next digested with BamHI to yield twoDNA fragments, both of which are ligated to the VZV fragment DNA to forma cosmid containing a 15-35 kb fragment of VZV genomic DNA.

To perform the ligation, approximately 3 μg of the SuperCos™ 1 vector(containing both BamHI fragments) was ligated to VZV DNA with AscIadaptors using ligase. Following ligation, the mixture was packaged intolambda phage particles using a commercially available packaging extract(Gigapack II packaging extract, Stratagene, La Jolla, Calif.). Thepackaged DNA was introduced into the E. coli strain XL1-MR (Stratagene,La Jolla, Calif.). Transfected bacteria were plated onto LB-ampicillinplates for selection and grown overnight at 37° C.

Individual ampicillin resistant colonies were picked and grown to latelog phase in LB-ampicillin broth. The cosmid DNA was isolated from thebacteria using techniques known in the art, digested with BamHI andelectrophoresed on an agarose gel. Each cosmid yielded a distinct andpredictable pattern. Two cosmid clones containing each restrictionfragment were identified and isolated. Their designations are asfollows:

Fsp1 (1-33211): pV Fsp 4 and pV Fsp 25

Spe1 (21875-62008): pV Spe 5 and pV Spe I4

Pme1 (53877-96188): pV Pme I9 and pV Pme 26

Spe1 (94208-124884): pV Spe 21 and pV Se 24

Large cultures of these cosmids were grown and the DNA isolated bymethods known in the art.

EXAMPLE 7

ORFS/L Gene Mutations and Cosmids

Cosmid clones containing mutant VZV ORFS/L genes were constructed usinga cosmid clone containing a wildtype VZV ORFS/L gene and a plasmid clonecontaining the PCR product of the entire ORSF/L gene as follows.

The first mutant VZV ORFS/L gene constructed was a deletion of nearlythe entire first reading frame of the ORFS/L gene ("first ORF") (aminoacids 99 through 223 of FIG. 6a were deleted), equal to approximately60% of the ORFS/L gene. The deletion mutant was constructed using the pVFsp4 cosmid which contains the VZV (Oka) nucleotide sequence(corresponding to VZV (Dumas) genomic nucleotides 1 through 33211) andcontains the first ORF of the ORFS/L gene. Accordingly, pV Fsp4 wasdigested with XhoI and PmeI to isolate a predicted 1190 nucleotide VZV(Oka) fragment (corresponding to VZV (Dumas) genomic nucleotides 24through 1214) which was then ligated into a pGEM7zf+ plasmid previouslydigested with XhoI and SacI. The 3' overhang of the SacI site was madeblunt ended by the addition of T4 DNA polymerase in order to facilitateligation. The resulting clone, containing the first ORF of the VZV (Oka)ORFS/L gene, was designated pV XLeft4 (containing nucleotidescorresponding to Dumas nucleotide sequence 24 through 1214).

In order to delete nearly all of the first ORF (the last 60% of the VZV(Oka) first ORF), pV XLeft4 was then partially digested with the enzymeApaLI. (Partial digestion was necessary to excise one fragment becauseApaLI cuts once within the VZV insert of pV XLeft4 (nucleotide 183 ofVZV(Dumas)) and twice within the pGEM7zf+ vector.) Following partialdigestion with ApaLI, the digested DNA was phenol/chloroform extractedto remove enzyme, the correct size fragment (containing both vector andthe first ORF) isolated and then digested to completion with BspEI.BspEI cuts at nucleotide 571 of the corresponding VZV (Dumas) nucleotidesequence. The BspEI site is less than 10 nucleotides downstream of theORFS/L gene termination codon in the first ORF. Thus, BspEI digestionreleases an approximately 388 bp fragment from the first ORF. Followingdigestion with BspEI, the two ends (ApaLI and BspEI) of the ApaL1/BspEIfragment (also containing the vector) were made blunt by the addition ofKlenow enzyme and the appropriate deoxynucleotides. The blunt ended DNAwas separated on an agarose gel, the appropriately sized fragmenteluted, ligated with itself and used to transform E. coli using methodsknown in the art. The resulting plasmid encompasses a deletion of thenucleotides between the ApaLI site within the ORFS/L gene and the BspEIsite downstream of ORFS/L. This plasmid was designated pV Left delta4and encodes VZV Oka nucleotides 225 through 383 where 383 is linked to772 through 1214 (nucleotide 1121 is the last sequenced nucleotide)(which correspond to the Dumas sequence nucleotides 24 through 183, and572 through 1214 where 183 is linked to 572).

The second mutant VZV ORFS/L gene constructed contained an insertion ofthe gB epitope encoding nucleotide sequence of Cytomegalovirus (CMV) atthe C-terminus of the first ORF. This mutant was constructed using theplasmid pG S/L C3 which contains the cloned, sequenced PCR product ofthe entire intact the VZV Oka ORFS/L nucleotide sequence (1121nucleotides in length). Accordingly, pG S/L C3 was digested with EcoRIand XbaI to remove the nucleotide sequence encoding the last 37C-terminal amino acids of the ORFS/L gene and to create a site forinserting a double stranded oligonucleotide encoding the gB epitope.(EcoRI cuts at nucleotide 444 of the corresponding VZV (Dumas) sequenceand XbaI cuts in the polylinker of the plasmid downstream from theORFS/L termination codon.) The resulting EcoRI/XbaI site provided aninsertion site for a double stranded oligonucleotide with EcoRI and XbaIsticky ends, codons for the 20 amino acid CMV gB epitope, a stop codonat the end of the epitope, and a KpnI site downstream of the terminationcodon. Two single stranded oligonucleotides, "S/L R1RevgB" and "S/LR1CodegB", with the following sequences:

CTAGGGTACC TTAGTGGCGA TATCCGTTCT TGCGGTGGCG GAGGCGGTCG AGGAGGTTGGGCTTCTGCCC CTT (S/L R1RevB) (SEQ ID NO:5), and

AATTAAGGGG CAGAAGCCCA ACCTCCTCGA CCGCCTCCGC CACCGCAAGA ACGGATATCGCCACTAAGGT ACC (S/L R1CodegB) (SEQ ID NO:6)

were annealed to make the double stranded oligonucleotide. The doublestranded oligonucleotide was ligated into the EcoRI/Xbal site and theresulting plasmid, pG ORFS/L CgB-7, was isolated using standardtechniques. The region of pG ORFS/L CgB-7 within and surrounding theoligonucleotide insertion was sequenced and confirmed to have thepredicted sequence. This plasmid, pG ORFS/L CgB-7, contains an ORFS/Lgene in which the nucleotides encoding the C-terminal 37 amino acids ofORFS/L are replaced by the nucleotides encoding the 20 amino acids ofthe CMV gB epitope (herein referred to as "ORFS/L-CgB insert").

The first ORF with its C-terminal 37 amino acids replaced with the gBepitope (herein referred to as "first ORF-CgB") was removed from theORFS/L-CgB insert of pG ORFS/L CgB-7 and subcloned into a pGEM7zf+vector for final insertion into a pV Fsp4 cosmid as follows. The firstORF-gB sequence of pG ORFS/L CgB-7 was excised by digestion with KpnIand XhoI (corresponding to nucleotide 24 to 444 of the Dumas nucleotidesequence, plus the 20 amino acid CMV gB epitope and the KpnI site). TheKpnI site in the excised fragment was converted to a blunt ended site bythe addition of T4 DNA polymerase and the appropriate nucleotides. Theblunt end KpnI/XhoI fragment was inserted into pV XLeft4 plasmid (thepGEM7zf+ vector containing the first ORF used for the deletion mutant)previously digested with BspEI (made blunt ended by the addition ofKlenow and the appropriate deoxynucleotides) and XhoI. This stepreplaced the deletion mutant insert, discussed above, with the firstORF-CgB sequence. The resulting plasmid with the first ORF-CgB sequenceand about 700 base pairs of VZV Oka DNA was designated pV XLeft4gB(corresponding to Dumas sequence nucleotides 24-444 linked to thenucleotide sequence encoding the CMVgB epitope and a blunt ended KpnIsite appended to nucleotides corresponding to Dumas sequence nucleotides572-1214, where the blunt ended KpnI site is linked to nucleotide 572).

The inserts from pV XLeft delta4 and pV XLeft4gB, first ORF deletionmutant and first ORF-CgB mutant (respectively), were cloned into VZVgenomic DNA contained within two separate pV Fsp4 cosmids as follows.The cosmid pV Fsp4 was digested to completion with XhoI (cuts atnucleotide 24 in the corresponding Dumas nucleotide sequence) and SgrA1(cuts at nucleotide 28743 in the corresponding Dumas nucleotidesequence) to release a 12 kilobase pair vector fragment containing thevector and about 5 kilobase pairs of VZV Oka DNA (corresponding tonucleotides 28,743 through 33,211 of the VZV Dumas nucleotide sequence).Fragments isolated using restriction enzymes XhoI (which cuts atnucleotide 24 in the corresponding Dumas nucleotide sequence) and SgrAI(which cuts at nucleotide 777 in the corresponding Dumas nucleotidesequence) from plasmids pV XLeft delta4 and pV XLeft4gB were insertedinto the corresponding sites of the 12kbp vector fragment. The resultingcosmids were designated pV dSgr d4 (containing the Oka nucleotidesequences corresponding to the Dumas nucleotide sequence 24 through 183linked to 572 through 1214, where nucleotide 183 is linked to nucleotide572) and pV dSgr 4gB (containing Oka nucleotide sequences correspondingto the Dumas nucleotide sequences 24 through 444 appended to nucleotidesencoding CMV gB epitope and blunt ended KpnI site, where the lastnucleotide in the blunt ended KpnI site is linked to 572 through 1214).

In order to replace the removed VZV DNA in cosmids pV dSgr d4 and pVdSgr 4gB, the SgrA1 fragment spanning the internal nucleotides (fromnucleotides 777 through 28743) was isolated by digestion of pV Fsp4 withSgrA1 and an agarose gel electrophoresis. This fragment was insertedinto either SgrA1 digested pV dSgr d4 or SgrA1 digested pV dSgr 4gB.Following ligation, the cosmids were packaged using a lambda packagingextract (Gigapack, Stratagene) and used to infect E. coli. The resultingcosmids, pV Fspdelta 4 and pV Fsp4gB, were identified by restrictionenzyme digestion. These two cosmids were then used to regenerate VZVthat contains either a deletion of the C-terminal 60% of ORFS/L or a VZVthat contains a gB epitope at the C-terminus of ORFS/L.

EXAMPLE 8

Transfection of MeWO Cells using the Four Cosmids Made with OverlappingWildtype VZV (Oka) DNA Fragments to Produce Reconstructed Wildtype VZV

The four cosmids prepared with overlapping VZV DNA fragments (pV Fsp4,pV Spe 5, pV Pme 19, and pV Spe21), as described above, were used totransfect MeWO cells and produce reconstructed wildtype VZV. As analternative to MeWO Cells, MRC-5, WI-38, human foreskin fibroblast cellscould be used with cosmids pV Fsp4, pV Spe5, pV Pme19, and pV Spe21 orother cosmids described herein containing mutant ORFS/L genes.

Prior to transfection, cosmids pV Fsp4, pV Spe5, pV Pme19 and pV Spe21,were prepared from E. coli using techniques known in the art anddigested with AscI to release the overlapping VZV AscI adapted fragmentsfrom each cosmid. AscI was chosen because there were no AscI sites foundin the VZV Oka strain. Approximately 10 μg of pV Fsp 4, pV Spe 5, and pVPme I9 and 5 μg of pV Spe21 cosmid DNA were separately digested withAscI. After digestion a small aliquot from each digest was removed toconfirm on agarose gel electrophoresis that digestion was complete. Theremainder of the digests, containing cosmid DNA and overlapping VZV AscIadapted fragments, were combined in a final solution of 10 mM EDTA toarrest AscI activity.

The combined cosmid DNA and overlapping VZV AscI adapted fragments("combined DNA") were co-purified by 3 extractions with equal volumes ofphenol and chloroform (chloroform is actually 24 parts chloroform and 1part iso-amyl alcohol) followed by one extraction with chloroform. Thecombined DNA was precipitated by the addition of sodium acetate to 0.3 Mand 3 volumes of ethanol. The combined DNA pellet was collected bycentrifugation for 10 minutes and then washed with 70% ethanol. Theethanol was removed and the combined DNA was resuspended in water,generally at 350 ng/μl (total combined DNA content).

For transfection of MeWO cells, at least 3 μg of combined DNA (mixed asdescribed above) and 0 to 500 ng of the plasmid pMS62 were diluted into250 μl of a CaCl₂ solution with a final concentration of 0.252 M. (Theplasmid pMS62 is a plasmid that contains VZV ORF62 under transcriptionalcontrol of the CMV major immediate early promoter. This plasmidincreases the reliability of generating infectious VZV using thistransfection system. See, Cohen, J. I, and Seidel, K. E., Proc. Natl.Acad. Sci. 90:7376-7380 (1993). The combined DNA/CaCl₂ solution wasslowly added to an equal volume of Hepes buffered saline (280 mM NaCl;10 mM KCl; 1.4 mM Na₂ HPO₄, 5.6 mM glucose; 20 mM HEPES, pH 7.05) toform a precipitate which was then incubated in the buffer for 30 minutesat room temperature. After aspiration to remove culture media, thecombined DNA precipitates were added to cells plated on an approximately25 cm² surface area for 500 μl of combined DNA solution. (The MeWO cellswere trypsinized 24 hours prior to the transfection procedure and seededwith 2×10⁶ MeWO cells per 25 cm².) Culture media was placed on the cellsand they were returned to an incubater for 2 to 6 hours. At this time,the cells were subjected to a glycerol shock as known in the art. Inbrief, 3 mls of 15% glycerol in Hepes buffered saline was added for 3min at 37° C. The glycerol solution was removed, the cells rinsed oncewith media and 5 ml of media was added. The cells were then assayed forplaque development after growth for 3-5 days. Optionally, no glycerolshock was performed and the cells were assayed for plaque developmentafter growth for 3-5 days.

The MeWO cells were assayed for plaque formation as follows: Three tofive days post-transfection, the cells are trypsinized to release thecells from the culture dish and reseeded in a vessel containing 3 timesmore surface area than the culture dish. Three to six days following thetrypsinization, the larger vessels were monitored for plaque formationwith a light microscope that allows visual scoring of plaques.Alternatively, the cells can be stained by rinsing twice with water andplaques can be visualized with a light microscope and counted moreaccurately compared to non stained cells.

Using this assay method, the disclosed method of reconstructing VZVproduced 250 plaques of reconstructed VZV using 3.5 μg of total cosmidDNA with 50 ng of pMS62; 450 plaques of reconstructed VZV using 3.5 μgof total cosmid DNA with 500 ng of pMS62; greater than 1,000 plaques(nearly confluent) reconstructed VZV using 7.0 μg of DNA cosmid DNA with50 ng of pMS62. The ratio of cosmids used with overlapping fragments(for total cosmid DNA) was 1:1:1:0.5 (pV Fsp4, pV Spe5, pV Pme 19 and pVSpe21, respectively).

In addition to making reconstructed wild type Oka VZV, this techniquecan be used with ORFS/L gene fragments produced from deletions, such as,but not limited to, the ApaL deletion described in Example 7, or ORFS/Lgene mutations as discussed herein, to make reconstructed mutant VZV.

EXAMPLE 9

Northern Analysis of RNA from VZV (Oka) Infected Cells Demonstrates thePresence of a ORFS/L Gene Transcript

Human foreskin fibroblast (HF) cells infected with wildtype VZV (Oka)were tested for the presence of an ORFS/L gene transcript follows. PCRwas used with the primer pair "ORFS/LPr2" and ORFS/LPr1" on DNA isolatedfrom the cytoplasm of VZV (Oka) infected HF cells to make a probefragment. Primer ORFS/LP 1 had the following sequence:

5'-GCCGCCATGGGATGAAAAAAGTGTCTGTCTGTCTGTGCG-3' SEQ ID NO: 7

Primer ORFS/LP 2 had the following sequence:

5'-GCCGCC ATGGTCATGTAGTTGAGTTGGGAGGTTCC-3' SEQ ID NO: 8

PCR was performed using these two primers in accordance with themanufacturer's instructions (Gene Amp, Perkin-Elmer).

A DNA fragment ("PCR DNA") of the appropriate size was generated fromthe PCR. The PCR DNA was digested with NcoI (the site had beenincorporated into the 5' region of both ORFS/LPr1 and ORFS/LPr2) andinserted into NcoI digested plasmid pGEM5zf+. The resulting plasmid wasdesignated pORFS/L C7. The vector pGEM5zf+ contains the phage Sp6 and T7RNA promoters on either side of the NcoI site.

Two different synthetic runoff ³² P-labeled RNA transcripts were madefrom pORFS/L C7 to use as probes of HF cell RNA.

The first probe was designated Sp6/EcoRI. pORFS/L C7 was digested withEcoRI and transcribed using Sp6 RNA polymerase and |³² P|CTP accordingto the manufacturer's instructions (Promega, Madison, Wis.). The ³² Plabeled RNA that was generated was the appropriate size (approximately650 nucleotides) and predicted to be the same sense as an ORFS/Ltranscript.

The second probe was designated T7/Xho. pORFS/L C7 was digested withXhoI and transcribed using T7 RNA polymerase and |³² P|CTP according tothe manufacturer's instructions (Promega, Madison, Wis.). The ³² Plabeled RNA that was generated was the appropriate size (approximately580 nucleotides) and predicted to be complementary to an ORFS/Ltranscript.

The RNA used in these experiments was from HF cells. Six 850 cm² rollerbottles of confluent HF cells were infected with VZV (Oka). 3 days postinfection the total RNA was harvested from the cells following themethod of Chomczymunski and Sacchi, Anal. Biochem. 162:156 (1987). 3roller bottles of uninfected HF were processed in parallel as controlRNA.

Polyadenylated RNA was also made from total HF RNA. Approximately 1 mgof the harvested RNA was enriched for polyadenylated RNA by affinitychromatography on oligo dT cellulose (Boehringer Mannheim). Nonspecificspecies were washed from the column with a 20 mM Tris (pH7.5); solutioncontaining 1 M LiCl, 0.2% SDS, and 2 mM EDTA. Specifically bound polyARNA was eluted with 10 mM Tris (pH 7.5), 1 mM EDTA, 0.0-5% SDS.

Total and polyadenylated RNA from infected and uninfected cells wasseparated by size on a 1% agarose/6.6% formaldehyde gel usingelectrophoresis. The following samples were loaded onto the gel.

1. 20 μg VZV infected HF whole cell RNA.

2. 20 μg VZV uninfected HF whole cell RNA.

3. 1 μg of VZV infected HF poly A enriched RNA.

4. 1 μg of VZV infected HF poly A depleted RNA.

5. 1 μg of uninfected HF poly A enriched RNA.

6. 1 μg of uninfected HF poly A depleted RNA.

Following electrophoresis, these samples were transferred from the gelto Hybond N+ paper by capillary action, as a blot. The nucleic acidswere then fixed to the Hybond by UV irradiation.

Duplicate samples of the RNA samples of the above gels and blots weremade. One blot was incubated with the Sp6/EcoRI (same sense) probe as acontrol and its duplicate incubated with the T7/XhoI (complementary)probe. The hybridization conditions were: 6×SSC; 1/×Denhardts; 30%foramide; 0.1 mg/ml single stranded sheared salmon sperm DNA at 58° C.for 17 hours. Unhybridized probe was washed off with 1.0×SSC; 0.1% SDSat 58° C. for 15 min followed by 0.1×SSC; 0.1% SDS at 58° C. for 15 min.In order to increase the specificity of the probe that remained bound,the filters were then washed 3 times in 2×SSC, incubated in 1 μg/mlRNAse A in 2×SSC for 15 min at room temperature, followed by a finalwash in 0.1×SSC; 0.1% SDS at 50° C. for 45 minutes. The washed,hybridized blots were exposed to X-ray film and developed at variousintervals.

The washed, hybridized blots yielded the following results:

    ______________________________________                                        HF RNA Sample  Sp6/EcoRI Probe                                                                           T7/XhoI Probe                                      ______________________________________                                        VZV Whole Cell --          +                                                    Uninfected Whole Cell -- --                                                   VZV Poly A enriched -- +                                                      VZV Poly A depleted -- --                                                     Uninfected Poly A -- --                                                       enrich                                                                        Uninfected poly A -- --                                                       depleted                                                                    ______________________________________                                    

An "-" indicates no band was observed and a "+" indicates a band wasobserved. The analysis indicated that a polyadenylated RNA ofapproximate 780 nucleotides was present in VZV (Oka) infected HF.Because the T7/XhoI probe is antisense to the predicted ORFS/L mRNA, theRNA that is being detected in these cells is the same sense as thepredicted ORFS/L mRNA. A shorter probe that was derived entirely fromthe C-terminal region of ORFS/L and used to determine the size of ORFS/Lgene transcripts in Northern blots also produced results consistent withthis result (data not shown). Because of the location of the openreading frames in VZV, ORFS/L is the only good candidate to be encodedby this transcript. The transcript size of the ORFS/L gene was 800-900bases in length.

EXAMPLE 10

Expression of ORFS/L (Oka) Proteins

To express different ORFS/L proteins from the ORFS/L gene of Oka thefollowing plasmids, described herein, were used:

pV4L (genomic clone encoding a 157 amino acid ORFS/L protein),

pG S/L C3 (PCR clone encoding a 223 amino acid ORFS/L protein), and

pG ORFS/L-CgB7 (PCR clone mutant encoding a 205 amino acid ORFS/Lprotein with 37 C-terminal amino acids replaced with 20 amino acids fromthe CMV gB epitope).

The plasmids were used with a cell free transcription/translation systemby Promega (TnT T7 Coupled Reticulocyte Lysate System). All reactionswere carried out according to the manufacturers directions. Plasmid DNA,used for the these experiments, was prepared according to standardtechniques known in the art. RNA was prepared in the complextranscription/translation reactions reaction using either T7 or SP6 RNApolymerase as indicated. The transcription/translation reactionsincluded, in addition to the above test plasmids, a number of negativeand positive controls to ensure that the expression system was workingcorrectly. The controls used were the luciferase control plasmid(supplied by Promega), no DNA, antisense DNA for the entire ORFS/L genefrom the PCR clone, and the γ₁ 34.5 gene from herpes simplex virus (pGICP 34.5).

In addition, the translation products of the ORFS/L gene from plasmidspG S/L C3 and pG ORF S/LCgB-7 were subjected to immunoprecipitationusing a mouse monoclonal antibody that reacts with the CMV gB epitope.Following in vitro transcription/translation of reactions containing thetwo plasmids pG S/L C3 and pG ORF S/LCgB-7, aliquots were separatelyremoved and the extracts were separately incubated with a mousemonoclonal antibody that reacts with the CMV gB epitope. This antibody,#1201, was purchased from the Goodwin Institute for Cancer Research,Inc. (Other designation for this antibody found in the literature isCH28). 1 ul of the antibody was incubated with each extract from the twotranscription/translation reactions for 30 minutes at room temperature.10 ul of a 50% slurry of CL4B sepharose-crosslinked protein A was addedto each antibody incubation and further incubated for 30 minutes at roomtemperature. Each sepharose sample was pelleted by a 10 second spin in amicrofuge and washed after successive spins as follows: twice in 0.6MNaCl/50 mM Tris pH 7.5 (500 ul), once in 0.1% SDS/0.05% NP40/10 mM TrispH 8.0/0.3M NaCl (500 ul), once in 0.6 M NaCl/50 mM Tris pH 7.5 (500ul), once in 0.3M NaCl/50 mM Tris pH 7.5 (500 ul), and once in 20 mMTris pH 8.3 (500 ul). Each pellet was then resuspended in 10 ul of H₂ O.

Samples from each transcription/translation reaction and samples fromeach immunoprecipitation were then subjected to gel electrophoresis asfollows. 10 ul of sample buffer was added to 10 ul of each sample(Sample buffer is 100 mM

DTT, 125 mM Tris (pH 7.0) 15% sucrose 4% SDS, 1 mM EDTA and 0.01%bromophenol blue). The samples were heated at 90° C. for 5 minutes andloaded onto a discontinuous SDS-polyacrylamide gel (a 12.5% resolvinggel) and electrophoresed at constant current. Following electrophoresis,the gel was fixed with 25% isopropanol and 10% acetic acid for 30minutes, incubated with the fluor solution AMPLIFY (purchased fromAmersham), and dried onto Whatmann paper. The gel was exposed toHyperfilm MP (Amersham) and machine developed.

The results of the transcription/translation reactions and theimmunoprecipitations are shown in FIG. 8. The lanes in FIG. 8 containthe following samples:

1. Luciferase positive control plasmid using T7 RNA polymerase,

2. No DNA using T7 RNA polymerase and Sp6 RNA polymerase,

3. pG S/L C3 (entire ORFS/L C3) using Sp6 RNA polymerase,

4. pG S/L C3 antisense control (entire ORFS/L C3) using T7 RNApolymerase,

5. pG ORFS/L-CgB-7 (ORFS/L protein with CMV epitope) using Sp6 RNApolymerase,

6. pV4L (first ORF) using T7 RNA polymerase,

7. pG ICP34.5 (herpes simplex viral protein) using Sp6 RNA polymerase,

8. pG S/L C3 (subjected to immunoprecipitation) using Sp6 RNApolymerase, and

9. pG ORFS/L-CgB-7 (subjected to immunoprecipitation) using Sp6 RNApolymerase.

The Sp6 transcripts from pG S/L C3, pG ORF S/L-CgB7, and pG ICP34.5generated proteins corresponding to ORF S/L (41kd and 28 kd bands), ORFS/L-gB (32 kd band), and γ₁ 34.5 (42 kd band) proteins, respectively.The T7 transcripts of the Luciferase control plasmid and pV4L generatedproteins correspond to firefly luciferase (62kd) and ORFS/L proteininitiated at the ATG (288 kd band), respectively. The T7 RNA generatedfrom pG S/L C3 was an antisense orientation of the ORF S/L gene and oldprotein encode an ORFS/L .

EXAMPLE 11

Testing for VZV Growth in Cell of Non-Neuronal Tissue Origin andAttenuated Neurovirulence

The reconstructed VZV and reconstructed mutant VZV of examples 7 and 8can be tested for retention of VZV growth properties and efficientreplication of the wild type VZV in cells from non-neuronal tissues andattenuated neurovirulence.

These properties can be assayed in cell culture. The γ₁ 34.5 deletionmutant of herpes simplex virus does not grow as well as the parentalvirus in the neuroblastoma cell line SK-N-SH as in J. Chou and B.Roizman, Proc. Natl. Acad. of Sci. (1992) 84:3266-3270, the methods ofwhich are herein incorporated by reference. A modified version of thistest is as follows.

The ability of a mutation to affect viral growth in neuronal tissuecells is determined using plague assays to measure VZV propagation.Growth curves of wild type VZV (Oka) and the modified VZV compositionsof the present invention are generated and compared by measuringinfective titers at various times post-infection using standard viralplaque forming assays known in the art. Mutant VZV is generated bycotransfecting MeWO cells with overlapping VZV fragments from cosmidclones as in examples 7 and 8. After cotransfection, infected cellsdevelop plaques and a stock of the virus is made by treating theinfected MeWO cells with trypsin to release the cells into the culturemedia. The cells are then used as an inoculum for human foreskinfibroblast (HF) cells. Separate HF cell cultures are infected with wildtype VZV and modified VZV of the present invention. After incubation for3 days at 34° C. trypsin is added to the HF cell cultures and viralstocks are prepared from each culture allocated and stored.

Aliquots of viral stock (wildtype or mutant VZV) are then used to infectcultures of "tester" cells. The tester cells include the humanneuroblastoma cell line SK-N-SH, or other human or murine neuroblastomacell lines. At designated points 1-5 days following infection, theneuroblastoma cells are subjected to treatment with trypsin and variousconcentrations of the trypsin treated cells are then added to aconfluent monolayer of MeWO cells, which form plaques after infection.Three to five days after infection, the MeWO cell monolayers infectedwith wild type or modified VZV infection reaches a maximum, the plaquesare counted and the virus titer is determined. The modified VZV titerwill be compared to the wild type virus titer in permissive cells (MRC-5or VERO cells) and "tester" cells. Thus, dual comparison provides anindication of the ability of the modified VZV to grow on the "tester"cells and permissive cells. Serum conditions can be adjusted to improvethe titer yield, See PCT application WO 93/24616. An ORFS/L genemutation decreases the plaque forming units (PFU) obtained from cells ofneuronal tissue origin. A mutation of the ORFS/L gene preferablydecreases the PFU at least 10 fold, more preferably at least 100 fold,and most preferably by at least 1,000 fold compared to the wild typeVZV.

Comparison of the titers and recombinant and parental virus will revealthe presence or absence of a growth defect in this cells line.

It is anticipated that mutant VZV will grow more poorly in SK-N-SH cellsthan the parental virus (wild type), yet will grow comparable to theparental virus in cells not of neuronal origin (such as VERO cells).Mutations of the ORFS/L gene that result in 30% slower growth inSK-N-SH, compared to the parent virus are preferred.

Biological Deposits

DNA samples containing four plasmids pG S/L C3, pV4L, pV21S, and pV 21Jwere deposited with American Type Culture Collection, 12301 ParlawnDrive, Rockville, Md. 20852 (ATCC Accession Number 75758, 75757, 75755and 75756 respectively).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  17                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Oligomer DNA                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCCGCCATGG AGGGGAGCGA CGGAACACG         - #                  - #                29                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Oligomer DNA                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GCCGCCATGG CTGTCGGCGG ACTATGAAC         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Oligomer DNA                                      - -   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - - GATCCGGCGC GCCA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: Oligomer DNA                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TGGCGCGCCG                - #                  - #                      - #        10                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Oligomer DNA                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CTAGGGTACC TTAGTGGCGA TATCCGTTCT TGCGGTGGCG GAGGCGGTCG  - #                  50                                                                         - - AGGAGGTTGG GCTTCTGCCC CTT           - #                  - #                    73                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Oligomer DNA                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - AATTAAGGGG CAGAAGCCCA ACCTCCTCGA CCGCCTCCGC CACCGCAAGA  - #                  50                                                                         - - ACCGATATCG CCACTAAGGT ACC           - #                  - #                    73                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Oligomer DNA                                      - -   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - - GCCGCCATGG GATGAAAAAA GTGTCTGTCT GTCTGTGCG      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Oligomer DNA                                      - -   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - - GCCGCCATGG TCATGTAGTT GAGTTGGGAG GTTCC       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  1126 ba - #se pairs                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (Genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 90...761                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AGAGTCGGGG GTGACGGAGT CCCCTCCTTT TCTCGTGAGC GCCACTGGCG CG -             #CGGACTGT     60                                                                 - - TTGTTGTTAA TAAAAGCGGA ACGGTTTTT ATG AAA AAA GTG TCT - # GTC TGT        CTG      113                                                                                      - #              Met Lys L - #ys Val Ser Val Cys Leu                         - #                1  - #             5                      - - TGC GGG CGG GCG ACG GGC GGG CTG GTC GGA CC - #C CCC CCC GAA AAT AAC          161                                                                       Cys Gly Arg Ala Thr Gly Gly Leu Val Gly Pr - #o Pro Pro Glu Asn Asn                10             - #     15             - #     20                          - - CCC CCC CCG GTT TCT GGG CGC CCG GCG GAC CC - #C GGG AGA GGA GGC CAG          209                                                                       Pro Pro Pro Val Ser Gly Arg Pro Ala Asp Pr - #o Gly Arg Gly Gly Gln            25                 - # 30                 - # 35                 - # 40       - - CCC TCT CGC GGC CCC CTC GAG AGA GAA AAA AA - #A AAG CGA CCC CAC CTC          257                                                                       Pro Ser Arg Gly Pro Leu Glu Arg Glu Lys Ly - #s Lys Arg Pro His Leu                            45 - #                 50 - #                 55              - - CCC GCG CGT TTG CGG GGC GAC CAT CGG GGG GG - #A TGG GAT TTT TTG CCG          305                                                                       Pro Ala Arg Leu Arg Gly Asp His Arg Gly Gl - #y Trp Asp Phe Leu Pro                        60     - #             65     - #             70                  - - GGA AAC CCC CCC CCG CCA GCC TTT AAC AAA AC - #C CGC GCC TTT TGC GTC          353                                                                       Gly Asn Pro Pro Pro Pro Ala Phe Asn Lys Th - #r Arg Ala Phe Cys Val                    75         - #         80         - #         85                      - - CAC CCC TCG TTT ACT GCT CGG ATG GCG ACC GT - #G CAC TAC TCC CGC CGA          401                                                                       His Pro Ser Phe Thr Ala Arg Met Ala Thr Va - #l His Tyr Ser Arg Arg                90             - #     95             - #    100                          - - CCT GGG ACC CCG CCG GTC ACC CTC ACG TCG TC - #C CCC AGC ATG GAT GAC          449                                                                       Pro Gly Thr Pro Pro Val Thr Leu Thr Ser Se - #r Pro Ser Met Asp Asp           105                 1 - #10                 1 - #15                 1 -      #20                                                                              - - GTT GCG ACC CCC ATC CCC TAC CTA CCC ACA TA - #C GCC GAG GCC GTG        GCA      497                                                                    Val Ala Thr Pro Ile Pro Tyr Leu Pro Thr Ty - #r Ala Glu Ala Val Ala                          125  - #               130  - #               135              - - GAC GCG CCC CCC CCT TAC AGA AGC CGC GAG AG - #T CTG GTG TTC TCC CCG          545                                                                       Asp Ala Pro Pro Pro Tyr Arg Ser Arg Glu Se - #r Leu Val Phe Ser Pro                       140      - #           145      - #           150                  - - CCT CTT TTT CCT CAC GTG GAG AAT GGC ACC AC - #C CAA CAG TCT TAC GAT          593                                                                       Pro Leu Phe Pro His Val Glu Asn Gly Thr Th - #r Gln Gln Ser Tyr Asp                   155          - #       160          - #       165                      - - TGC CTA GAC TGC GCT TAT GAT GGA ATC CAC AG - #A CTT CAG CTG GCT TTT          641                                                                       Cys Leu Asp Cys Ala Tyr Asp Gly Ile His Ar - #g Leu Gln Leu Ala Phe               170              - #   175              - #   180                          - - CTA AGA ATT CGC AAA TGC TGT GTA CCG GCT TT - #T TTA ATT CTT TTT GGT          689                                                                       Leu Arg Ile Arg Lys Cys Cys Val Pro Ala Ph - #e Leu Ile Leu Phe Gly           185                 1 - #90                 1 - #95                 2 -      #00                                                                              - - ATT CTC ACC CTT ACT GCT GTC GTG GTC GCC AT - #T GTT GCC GTT TTT        CCC      737                                                                    Ile Leu Thr Leu Thr Ala Val Val Val Ala Il - #e Val Ala Val Phe Pro                          205  - #               210  - #               215              - - GAG GAA CCT CCC AAC TCA ACT ACA TGAAACTACT GT - #CCGGAAGG GGAAGGTATT         791                                                                       Glu Glu Pro Pro Asn Ser Thr Thr                                                           220                                                                - - TATTCTCGCT TGCAGCTTGT CGCGCGTGTA TGCACAACAA AAGCTATAAT AT -             #GTCACCAA    851                                                                 - - AGCCAACGTC GCCATCTGGA GTACTACACC CAGTACGTTG CATAACCTGT CC -            #ATTTGCAT    911                                                                 - - TTTCAGTTGC GCGGACGCCT TTCTCCGGGA TCGTGGCCTT GGGACATCAA CC -            #AGTGGAAT    971                                                                 - - AAGAACCGCC GGTGGTCTTG TTTGAACGAC GAGTGGCGAC GCGTTGTTCT GC -            #ATAAGCTC   1031                                                                 - - TGTATGCTGA TACATAAACA CAGAGTCTGT ATCGCTATCA GATTCCCGAA CA -            #CCTTCCGG   1091                                                                 - - TACCCCATAC TCCGATACCC TGGACATTGC GGATC       - #                       - #     1126                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 224 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: Protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Met Lys Lys Val Ser Val Cys Leu Cys Gly Ar - #g Ala Thr Gly Gly        Leu                                                                               1               5 - #                 10 - #                 15             - - Val Gly Pro Pro Pro Glu Asn Asn Pro Pro Pr - #o Val Ser Gly Arg Pro                   20     - #             25     - #             30                  - - Ala Asp Pro Gly Arg Gly Gly Gln Pro Ser Ar - #g Gly Pro Leu Glu Arg               35         - #         40         - #         45                      - - Glu Lys Lys Lys Arg Pro His Leu Pro Ala Ar - #g Leu Arg Gly Asp His           50             - #     55             - #     60                          - - Arg Gly Gly Trp Asp Phe Leu Pro Gly Asn Pr - #o Pro Pro Pro Ala Phe       65                 - # 70                 - # 75                 - # 80       - - Asn Lys Thr Arg Ala Phe Cys Val His Pro Se - #r Phe Thr Ala Arg Met                       85 - #                 90 - #                 95              - - Ala Thr Val His Tyr Ser Arg Arg Pro Gly Th - #r Pro Pro Val Thr Leu                  100      - #           105      - #           110                  - - Thr Ser Ser Pro Ser Met Asp Asp Val Ala Th - #r Pro Ile Pro Tyr Leu              115          - #       120          - #       125                      - - Pro Thr Tyr Ala Glu Ala Val Ala Asp Ala Pr - #o Pro Pro Tyr Arg Ser          130              - #   135              - #   140                          - - Arg Glu Ser Leu Val Phe Ser Pro Pro Leu Ph - #e Pro His Val Glu Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Thr Thr Gln Gln Ser Tyr Asp Cys Leu As - #p Cys Ala Tyr Asp        Gly                                                                                             165  - #               170  - #               175             - - Ile His Arg Leu Gln Leu Ala Phe Leu Arg Il - #e Arg Lys Cys Cys Val                  180      - #           185      - #           190                  - - Pro Ala Phe Leu Ile Leu Phe Gly Ile Leu Th - #r Leu Thr Ala Val Val              195          - #       200          - #       205                      - - Val Ala Ile Val Ala Val Phe Pro Glu Glu Pr - #o Pro Asn Ser Thr Thr          210              - #   215              - #   220                          - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  1121 ba - #se pairs                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AGTCGGGGGT GACGGAGTCC CCTCCTTTTC TCGTGASCCA CTGGCGCGCG GA -             #CTGTTTGT     60                                                                 - - TGTTTGTTAA TAAAAGCGGA ACGGTTTTTA TGAAAAAAGT GTCTGTCTGT CT -            #GTGCGGGC    120                                                                 - - GGGCGACGGG CGGGCTGGTC GGACCCCCCC CGAAAATAAC CCCCCCCGGT TT -            #CTGGGCGC    180                                                                 - - CCGGCGGACC CCGGGGGGGG GGCCCAGCCC TCTCGCGGCC CCCTCGAGAG AG -            #AAAAAAAA    240                                                                 - - AAGCGACCCC ACCTCCCCGC GCGTTTGCGG GGCGACCATC GGGGGGGATG GG -            #ATTTTTTG    300                                                                 - - CCGGGAAACC CCCCCCGCCA GCCTTTAACA AAACCCGCGC CTTTTGCGTC CA -            #CCCCTCGT    360                                                                 - - TTACTGCTCG GATGGCCACC GTGCACTACT CCCGCCGACC TGGGACCCCG CC -            #GGTCACCC    420                                                                 - - TCACGTCGTC CCCCGGCATG GATGACGTTG CGACCCCCAT TCCCTACCTA CC -            #CACATACG    480                                                                 - - CCGAGGCCGT GGCAGACGCG CCCCCCCCTT ACAGAAGCCG CGAGAGTCTG GT -            #GTTCTCCC    540                                                                 - - CGCCTCTTTT TCCTCACGTG GAGAATGGCA CCACCCAACA GTCTTACGAT TG -            #CCTAGACT    600                                                                 - - GCGCTTATGA TGGAATCCAC AGACTTCAGC TGGCTTTTCT AAGAATTCGC AA -            #ATGCTGTG    660                                                                 - - TACCGGCTTT TTTAATTCTT TTTGGTATTC TCACCCTTAC TGCTGTCGTG GT -            #CGCCATTG    720                                                                 - - TTGCCGTTTT TCCCGAGGAA CCTCCCAACT CAACTACATG AAACTACTGT CC -            #GGAAGGGA    780                                                                 - - AGGTATTTAT TCTGCTTGCA GCTTGTCGCG CGTGTATGCA CAACAAAAGC TA -            #TATATGTC    840                                                                 - - ACCAAAGCCA ACGTCGCCAT CTGGAGTACT ACACCCAGTA CATTGCATAA CC -            #TGTCCATT    900                                                                 - - TGCATTTTCA GTTGCGCGGA CGCCTTTCTC CGGGATCGTG GCCTTGGGAC AT -            #CAACCAGT    960                                                                 - - GGAATAAGAA CCGCCGGTGG TCTTGCCCGA ACGACGAGTG GCGACGCGTT GT -            #TCTGCATA   1020                                                                 - - AGCTCTGTAT GCTGATACAT AAACACAGAG TCTGTATCGC TATCAGATTC CC -            #GAACACCT   1080                                                                 - - TCCGGTACCC CATACTCCGA TACCCTGGAC ATTGCGGATC C    - #                      - # 1121                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 223 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: Protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Met Lys Lys Val Ser Val Cys Leu Cys Gly Ar - #g Ala Thr Gly Gly Leu      1               5   - #                10  - #                15               - - Val Gly Pro Pro Pro Lys Ile Thr Pro Pro Gl - #y Phe Trp Ala Pro Gly                  20      - #            25      - #            30                   - - Gly Pro Arg Gly Gly Gly Pro Ala Leu Ser Ar - #g Pro Pro Arg Glu Arg              35          - #        40          - #        45                       - - Lys Lys Lys Ala Thr Pro Pro Pro Arg Ala Ph - #e Ala Gly Arg Pro Ser          50              - #    55              - #    60                           - - Gly Gly Met Gly Phe Phe Ala Gly Lys Pro Pr - #o Pro Pro Ala Phe Asn      65                  - #70                  - #75                  - #80        - - Lys Thr Arg Ala Phe Cys Val His Pro Ser Ph - #e Thr Ala Arg Met Ala                      85  - #                90  - #                95               - - Thr Val His Tyr Ser Arg Arg Pro Gly Thr Pr - #o Pro Val Thr Leu Thr                  100      - #           105      - #           110                  - - Ser Ser Pro Gly Met Asp Asp Val Ala Thr Pr - #o Ile Pro Tyr Leu Pro              115          - #       120          - #       125                      - - Thr Tyr Ala Glu Ala Val Ala Asp Ala Pro Pr - #o Pro Tyr Arg Ser Arg          130              - #   135              - #   140                          - - Glu Ser Leu Val Phe Ser Pro Pro Leu Phe Pr - #o His Val Glu Asn Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Thr Gln Gln Ser Tyr Asp Cys Leu Asp Cy - #s Ala Tyr Asp Gly        Ile                                                                                             165  - #               170  - #               175             - - His Arg Leu Gln Leu Ala Phe Leu Arg Ile Ar - #g Lys Cys Cys Val Pro                  180      - #           185      - #           190                  - - Ala Phe Leu Ile Leu Phe Gly Ile Leu Thr Le - #u Thr Ala Val Val Val              195          - #       200          - #       205                      - - Ala Ile Val Ala Val Phe Pro Glu Glu Pro Pr - #o Asn Ser Thr Thr              210              - #   215              - #   220                          - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  561 bas - #e pairs                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (Genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GGCCCAGCCC TCTCGCGGCC CCCTCGAGAG AGAAAAAAAA AAGCGACCCC AC -             #CTCCCCGC     60                                                                 - - GCGTTTGCGG GGCGACCATC GGGGGGGATG GGATTTTTTG CCGGGAAACC CC -            #CCCCGCCA    120                                                                 - - GCCTTTAACA AAACCCGCGC CTTTTGCGTC CACCCCTCGT TTACTGCTCG GA -            #TGGCCACC    180                                                                 - - GTGCACTACT CCCGCCGACC TGGGACCCCG CCGGTCACCC TCACGTCGTC CC -            #CCGGCATG    240                                                                 - - GATGACGTTG CGACCCCCAT TCCCTACCTA CCCACATACG CCGAGGCCGT GG -            #CAGACGCG    300                                                                 - - CCCCCCCCTT ACAGAAGCCG CGAGAGTCTG GTGTTCTCCC CGCCTCTTTT TN -            #CTCACGTG    360                                                                 - - GAGAATGGCA CCACCCAACA GTCTTACGAT TGCCTAGACT GCGCTTATGA TG -            #GAATCCAC    420                                                                 - - AGACTTCAGC TGGCTTTTCT AAGAATTCGC AAATGCTGTG TACCGGCTTT TT -            #TAATTCTT    480                                                                 - - TTTGGTATTC TCACCCTTAC TGCTGTCGTG GTCGCCATTG TTGCCGTTTT TC -            #CCGAGGAA    540                                                                 - - CCTCCCAACT CAACTACATG A           - #                  - #                     561                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 157 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: Protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Met Gly Phe Phe Ala Gly Lys Pro Pro Pro Pr - #o Ala Phe Asn Lys Thr        1               5 - #                 10 - #                 15              - - Arg Ala Phe Cys Val His Pro Ser Phe Thr Al - #a Arg Met Ala Thr Val                   20     - #             25     - #             30                  - - His Tyr Ser Arg Arg Pro Gly Thr Pro Pro Va - #l Thr Leu Thr Ser Ser               35         - #         40         - #         45                      - - Pro Gly Met Asp Asp Val Ala Thr Pro Ile Pr - #o Tyr Leu Pro Thr Tyr           50             - #     55             - #     60                          - - Ala Glu Ala Val Ala Asp Ala Pro Pro Pro Ty - #r Arg Ser Arg Glu Ser       65                 - # 70                 - # 75                 - # 80       - - Leu Val Phe Ser Pro Pro Leu Phe Pro His Va - #l Glu Asn Gly Thr Thr                       85 - #                 90 - #                 95              - - Gln Gln Ser Tyr Asp Cys Leu Asp Cys Ala Ty - #r Asp Gly Ile His Arg                  100      - #           105      - #           110                  - - Leu Gln Leu Ala Phe Leu Arg Ile Arg Lys Cy - #s Cys Val Pro Ala Phe              115          - #       120          - #       125                      - - Leu Ile Leu Phe Gly Ile Leu Thr Leu Thr Al - #a Val Val Val Ala Ile          130              - #   135              - #   140                          - - Val Ala Val Phe Pro Glu Glu Pro Pro Asn Se - #r Thr Thr                  145                 1 - #50                 1 - #55                            - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 222 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (Genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GGCGGACTGT TTGTTGTTTG TTAATAAAAG CGGAACGGTT TTTATGAAAA AA -             #GTGTCTGT     60                                                                 - - CTGTCTGTGC GGGCGGGCGA CGGVCGGGCT GGTCGGACCC CCCCCGAAAA TA -            #ACCCCCCC    120                                                                 - - CGGTTTCTGG GCGCCCGGCG GACCCCGGGG GGGGGGGCCC AGCCCTCTCG CG -            #GCCCCCTC    180                                                                 - - GAGAGAGAAA AAAAAAAGCG ACCCCACCTC HYCGCGCGTT TG    - #                      - # 222                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 207 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (Genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - GTCAGAGTCG GGGGTGACGG AGTCCCCTCC TTTTCTCGTG AGCGCCACTG GC -             #GCGCGGAC     60                                                                 - - TGTTTGTTGT TTGTTAATAA AAGCGGAACG GTTTTTATGA AAAAAGTGTC TG -            #TCTGTCTG    120                                                                 - - TGCGNGCGGG CGACGGGCGG GCTGGTCGGA CCCCCCCCGA AAATAACCCC CC -            #CCGGTTTC    180                                                                 - - TGGGCGCCCG GCGGACCCCG GGGGGGG          - #                  - #                207                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 716 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (Genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - GGCGGACTGT TTGTTGTTTG TTAATAAAAG CGGAACGGTT TTTATGAAAA AA -             #GTGTCTGT     60                                                                 - - CTGTCTGTGC GGGCGGGCGA CGGVCGGGCT GGTCGGACCC CCCCCGAAAA TA -            #ACCCCCCC    120                                                                 - - CGGTTTCTGG GCGCCCGGCG GACCCCGGGG GGGGGGGCCC AGCCCTCTCG CG -            #GCCCCCTC    180                                                                 - - GAGAGAGAAA AAAAAAAGCG ACCCCACCTC CCCGCGCGTT TGCGGGGCGA CC -            #ATCGGGGG    240                                                                 - - GGATGGGATT TTTTGCCGGG AAACCCCCCC CGCCAGCCTT TAACAAAACC CG -            #CGCCTTTT    300                                                                 - - GCGTCCACCC CTCGTTTACT GCTCGGATGG CCACCGTGCA CTACTCCCGC CG -            #ACCTGGGA    360                                                                 - - CCCCGCCGGT CACCCTCACG TCGTCCCCCG GCATGGATGA CGTTGCGACC CC -            #CATTCCCT    420                                                                 - - ACCTACCCAC ATACGCCGAG GCCGTGGCAG ACGCGCCCCC CCCTTACAGA AG -            #CCGCGAGA    480                                                                 - - GTCTGGTGTT CTCCCCGCCT CTTTTTNCTC ACGTGGAGAA TGGCACCACC CA -            #ACAGTCTT    540                                                                 - - ACGATTGCCT AGACTGCGCT TATGATGGAA TCCACAGACT TCAGCTGGCT TT -            #TCTAAGAA    600                                                                 - - TTCGCAAATG CTGTGTACCG GCTTTTTTAA TTCTTTTTGG TATTCTCACC CT -            #TACTGCTG    660                                                                 - - TCGTGGTCGC CATTGTTGCC GTTTTTCCCG AGGAACCTCC CAACTCAACT AC - #ATGA            716                                                                     __________________________________________________________________________

I claim:
 1. An isolated ORFS/L gene comprising a nucleic acid sequenceselected from the group consisting of the sequences enumerated in SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13; SEQ ID NO:15, SEQ ID NO:16 and SEQ IDNO:17.
 2. A vector comprising a sequence according to claim
 1. 3. Avector selected from the group consisting of pV21S, pV21J, pV4L and pGS/L C3 deposited in the American Type Culture Collection as accessionnumbers 75755, 75756, 75757 and 75758 respectively.
 4. A cell infectedwith a vector according to claim
 2. 5. A cell according to claim 4wherein said cell is a VERO, HF, SK-N-SH or MeWo cell.
 6. A mutantORFS/L gene having a mutation that is not present in an ORFS/L gene of agenome of a naturally occurring VZV strain, said mutation(1) comprisinga deletion of at least 30 nucleotides in said ORFS/L gene, and (2)resulting in attenuated neurovirulence of VZV strains possessing saidmutant ORFS/L gene as compared to naturally occurring VZV strains.
 7. Amutant ORFS/L gene according to claim 6, wherein said mutation comprisesa deletion of a nucleotide sequence encoding amino acids 99 through 223of the ORFS/L gene as shown in SEQ ID NO:12.
 8. A mutant ORFS/L geneaccording to claim 6, wherein said mutation comprises a deletion of allcoding sequence of the ORFS/L gene.
 9. A mutant ORFS/L gene according toclaim 6, wherein said deletion comprises a deletion of the nucleotidesbetween the ApaL1 site ad the BspEI site of the ORFS/L gene.
 10. Amutant ORFS/L gene according to claim 6 wherein said mutationcomprises(1) a deletion of a nucleotide sequence encoding amino acids121 through 157 of the ORFS/L gene as shown in SEQ ID NO:14, and (2)replacement of said sequence with a heterologous nucleotide sequencewhich encodes 20 amino acids of a viral protein not encoded by a genomeof a naturally occurring VZV strain.
 11. A mutant ORFS/L gene accordingto claim 10, wherein said viral protein is a CMV gB protein.
 12. Amutant ORFS/L gene having a mutation that is not present in an ORFS/Lgene of a genome of a naturally occurring VZV strain, wherein saidmutation comprises a insertion of a heterologous nucleotide sequencewhich introduces a stop codon in the first ORF of said mutant ORFS/Lgene.